Antibodies which bind type i cannabinoid receptor/angiotensis ii receptor heteromers

ABSTRACT

Compounds and compositions useful for the treatment of liver diseases and methods of treating liver diseases are disclosed. The compounds of the invention specifically interact with heteromers of cannabinoid receptors as compared to monomers or homodimers. The invention also relates to methods of screening for compounds useful for the treatment of liver diseases and to methods of screening for diacylglycerol lipase inhibitors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/839,236, filed Dec. 12, 2017, now abandoned, which is a continuationof U.S. patent application Ser. No. 14/955,586, filed Dec. 1, 2015, nowU.S. Pat. No. 9,855,290, which is a continuation of U.S. patentapplication Ser. No. 13/575,220, filed Mar. 18, 2013, now U.S. Pat. No.9,216,220, which is the U.S. National Application under 35 U.S.C. § 371of International Patent Application No. PCT/US2011/022452, filed Jan.25, 2011 which claims priority to U.S. Provisional Application No.61/297,895, filed Jan. 25, 2010, the entire contents of which are herebyincorporated by reference.

GOVERNMENT SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under DA008863 awardedby The National Institutes of Health. The government has certain rightsin the invention.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The present specification is filed with a computer readable form (CRF)copy of the Sequence Listing. The CRF entitled27527_0070004_SEQ_LISTING.txt, which was created on Aug. 22, 2019 and is2,382 bytes in size, is identical to the paper copy of the SequenceListing and is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention is related to compounds and compositions usefulfor the treatment of liver diseases and related screening andtherapeutic methods. The present invention also relates to methods ofscreening for diacylglycerol lipase inhibitors.

BACKGROUND OF THE INVENTION

A fundamental question in liver biology is how type I cannabinoidreceptors (CB₁Rs) mediate liver fibrosis. Cannabinoid receptors are aclass of G-protein coupled receptors (GPCRs). Cannabinoid receptorsexhibit marginal expression in the normal liver but undergo enhancedexpression in the fibrotic human liver, predominately in activatedhepatic stellate cells (HSCs)². A critical aspect in chronic liverdisease is the activation of resident HSCs into proliferative,contractile, and fibrogenic cells. These myofibroblasts produce anexcess of extracellular matrix proteins including collagens, resultingin fibrosis. In these cells, specific mitogenic signaling cascades, uponactivation of naturally occurring GPCRs such as AT1R and adenosine 2Areceptor (A2aR) contribute to the fibrosis. HSCs are the cells primarilyresponsible for the fibrogenic response in the liver¹. Preventing theexpression or activation of CB₁Rs attenuates the development offibrosis². CB₁Rs contribute to fibrogenic response, since administrationof the CB₁R antagonist Rimonabant (SR141716) or genetic ablation of CB₁Rinhibits fibrosis progression in three distinct models of chronic liverinjury (namely, CCl₄-, thioacetamide-, and bile duct ligation-inducedfibrosis)². However, the extent of the contribution of CB₁Rs inalcohol-induced liver injury, and the molecular mechanism by which CB₁Rspromote liver fibrosis are not understood.

Approximately 25,000 people die each year of cirrhosis in the UnitedStates. The leading causes of cirrhosis are viral hepatitis, alcoholismand obesity-related liver diseases. Close to 1 million people sufferfrom this disease, which is the 7th leading cause of death in the UnitedStates, and whose only effective treatment is liver transplantation. Itis known that cannabinoid receptors are upregulated in the liver duringliver fibrosis, and the blockade of the CB₁ subtype of the receptor hasbeen shown to inhibit fibrogenesis, offering an approach for thetreatment of cirrhosis. However, as CB₁R expression is not localizedspecifically to the liver (in fact, CB₁Rs are likely the most widelyexpressed GPCR in the brain), the treatment of liver fibrosis andcirrhosis by the administration of a CB₁R inhibitor would like result insignificant side effects. In fact, the targeting of CB1 receptor in thetreatment of obesity has posed serious safety problems associated withsevere side effects. For example, (1) the Sanofi-Aventis CB₁R antagonistRimonabant (SR141716) was taken off the market because of its seriousside effects; (2) Merck discontinued clinical trials with its CB₁Rinverse agonist Taranabant (MK-0364) due to the high level of centralside effects, mainly depression and anxiety; and (3) Bristol MyersSquibb also discontinued development of its CB₁R antagonist Otenabant(CP-945,598) following the problems seen during clinical use of thesimilar drug Rimonabant. Therefore, what are needed are specificantagonists that do not have these systemic, toxic side effects.

N-arachidonoyl-ethanolamine (AEA, anandamide) and 2-arachidonoylglycerol (2-AG) are the two most studied endocannabinoids. They arebiosynthesized by cleavage of their membrane lipid precursorsN-arachidonoyl-phosphatidylethanolamine andsn-1-acyl-2-arachidonoylglycerols (DAGs) respectively, and theninactivated by intracellular hydrolyzing enzymes. The biologicalactivity of AEA and 2-AG is mainly mediated by activation of thecannabinoid receptors CB₁R and CB₂R.

2-AG has a variety of effects in vivo; it is a mediator of neuriteoutgrowth, during brain development, or as retrograde signal mediatingdepolarization-induced suppression of neurotransmission and synapticplasticity, in the adult brain. It protects neurons from inflammation bypreventing Cox2 gene expression (D2). In addition, 2-AG is involved inmetabolic regulation and diseases. For example, it is involved in boneformation (D3), in obesity (its concentration is increased in severaltissues and in the circulation of obese persons (D4)). 2-AG is highlyupregulated during chronic liver diseases and is implicated in thepathogenesis of non-alcoholic fatty liver disease, progression offibrosis to cirrhosis and the development of the cardiovascularabnormalities of cirrhosis, such as the hyperdynamic circulatorysyndrome and cirrhotic cardiomiopathy (D5).

2-AG is produced from the hydrolysis of phosphoinositol bisphosphate(PIP2), catalyzed by the PIP2-selective phospholipase C, or from thehydrolysis of phosphatidic acid (PA), catalyzed by a PA phosphohydrolaseinto diacylglycerols (DAGs). DAGs are then converted into 2-AG by sn-1selective-DAG lipases (DAGLs). Two sn-1 DAG lipase isozymes (DAGLα andDAGLβ) have been cloned and enzymatically characterized (D6). They aremostly located in the plasma membrane, are stimulated by Ca²⁺ andglutathione, appear to possess a catalytic triad typical of serinehydrolases, and do not exhibit strong selectivity for2-arachidonate-containing DAGs (FIG. 1). Endocannabinoids, including2-AG, stimulate CB₁R, resulting, among other things, in the activationof the ERK1/2 pathway, and phosphorylation of ERK1/2.

Because AEA and 2-AG biosynthetic enzymes have been identified onlyrecently, little information on the development of selective inhibitorsfor these proteins is currently available. RHC80267[1,6-bis-(cyclohexyloximino-carbonylamino)-hexane], andtetrahydrolipstatin (THL), have been shown to inhibit DAGL atconcentrations lower than those required to inhibit other lipases (D6).Furthermore, two inhibitors of 2-AG biosynthesis have been developed sofar, O-3640 and O-3841, with excellent selectivity for DAGLα over theother proteins of the endocannabinoid system tested. However, they arenot suitable for in vivo use (D7). Therefore, the identification of newinhibitors of DAGLs, which could be used in vivo, is highly clinicallyrelevant.

SUMMARY OF THE INVENTION

This Summary is provided to present a summary of the invention tobriefly indicate the nature and substance of the invention. It issubmitted with the understanding that it will not be used to interpretor limit the scope or meaning of the claims.

Embodiments of the invention comprise an isolated monoclonal antibodywhich specifically binds to a type I cannabinoid receptor(CB₁R)/angiotensin II receptor (AT1R) heteromer. In another preferredembodiment, a pharmaceutical composition comprises the monoclonalantibody and a pharmaceutically acceptable carrier or diluent.

In another preferred embodiment, a method of treating a liver disease ina mammal, comprises administering to the mammal in need of suchtreatment an effective amount of a compound, that preferentiallyinhibits an activity of a type I cannabinoid receptor (CB₁R)/angiotensinII receptor (AT1R) heteromer, as compared to a control receptor selectedfrom the group consisting of CB₁R monomer, CB₁R homodimer, AT1R monomer,AT1R homodimer, and a combination thereof. Preferably, the activity ofthe CB₁R/AT1R heteromer is inhibited at least about 5-fold greater thanthe activity of the control receptor. Preferably the mammal is human.

In a preferred embodiment, the activity is determined by measuringphosphorylation of extracellular signal-regulated kinase-1 (ERK1) orextracellular signal-regulated kinase-2 (ERK2).

In another preferred embodiment, the liver disease is selected from thegroup consisting of liver fibrosis, a liver disease associated withobesity, a liver disease associated with metabolic syndrome, livercirrhosis, alcoholic fibrosis, non-alcoholic fibrosis, fatty liver,hepatic cirrhosis, a diabetes-associated liver disease, genetic liverdiseases, liver inflammation, liver steatosis, or chronic hepatitis.

In another preferred a method of treating a liver disease in a mammal,comprising administering to the mammal in need of such treatment aneffective amount of a compound, that preferentially inhibits an activityof a type I cannabinoid receptor (CB₁R)/adenosine 2a receptor (A2aR)heteromer, as compared to a control receptor selected from the groupconsisting of CB₁R monomer, CB₁R homodimer, A2aR monomer, and A2aRhomodimer. Preferably, the activity of CB₁R/A2aR heteromer is inhibitedat least about 5-fold greater than the activity of the control receptor.

In another preferred embodiment, the activity is determined by measuringphosphorylation of extracellular signal-regulated kinase-1 (ERK1) orextracellular signal-regulated kinase-2 (ERK2).

In another preferred embodiment, a method of screening for a candidatecompound useful for the treatment of a liver disease, the methodcomprises a) contacting the compound with a CB₁R/A2aR heteromer, andwith at least one control receptor selected from the group consisting ofa CB₁R monomer, a CB₁R homodimer, an A2aR monomer, and an A2aRhomodimer, b) measuring inhibition of an activity of the CB₁R/A2aRheteromer and of the control receptor in the presence of the compound,and c) selecting the compound which preferentially inhibits the activityof the CB₁R/A2aR heteromer as compared to the control receptor, as acandidate compound useful for the treatment of the liver disease.Preferably, the wherein the candidate compound inhibits the activity ofthe CB₁R/A2aR heteromer at least about 5-fold greater than the activityof the control receptor.

In another preferred embodiment, the activity is determined by measuringphosphorylation of extracellular signal-regulated kinase-1 (ERK1) orextracellular signal-regulated kinase-2 (ERK2).

In another preferred embodiment, a method of screening for a compounduseful for the treatment of a liver disease, the method comprises a)contacting the compound with a CB₁R/AT1R heteromer, and with at leastone control receptor selected from the group consisting of a CB₁Rmonomer, a CB₁R homodimer, an AT1R monomer, and an AT1R homodimer; b)measuring inhibition of an activity of the CB₁R/AT1R heteromer and ofthe control receptor in the presence of the compound, and c) selectingthe compound which preferentially inhibits the activity of the CB₁R/AT1Rheteromer as compared to the control receptor, as a candidate compounduseful for the treatment of the liver disease. Preferably, the candidatecompound inhibits the activity of the CB₁R/AT1R heteromer at least about5-fold greater than the activity of the control receptor. In oneembodiment, the activity is determined by measuring phosphorylation ofextracellular signal-regulated kinase-1 (ERK1) or extracellularsignal-regulated kinase-2 (ERK2).

In another preferred embodiment, a method of screening for a compounduseful for the treatment of a liver disease, the method comprises a)contacting the compound with a cell expressing a type I cannabinoidreceptor (CB₁R)/angiotensin II receptor (AT1R) heteromer, and with acontrol cell expressing CB₁R alone or AT1R alone; b) measuringinhibition of endocannabinoid activity in the cell expressing CB₁R/AT1Rheteromer and in the control cell in the presence of the compound, andc) selecting the compound which preferentially inhibits endocannabinoidactivity in the cell expressing the CB₁R/AT1R heteromer as compared tothe control cell, as the candidate compound useful for the treatment ofthe liver disease.

In a preferred embodiment, the inhibition of endocannabinoid activity isdetermined by measuring angiotensin II-mediated ERK1/2 phosphorylation.Preferably, the inhibition of endocannabinoid activity in the cellexpressing the CB₁R/AT1R heteromer is at least about 5-fold greater thanthe inhibition of endocannabinoid activity in the control cell. In onepreferred embodiment, the cell expressing the CB₁R/AT1R heteromer, CB₁Ralone, or AT1R alone is HEK293 or CHO cell.

In another preferred embodiment, a method of screening for a compounduseful for the treatment of a liver disease, the method comprises a)contacting the compound with a cell expressing a type I cannabinoidreceptor (CB₁R)/adenosine 2a receptor (A2aR) heteromer, and with acontrol cell expressing CB₁R alone or A2aR alone; b) measuringinhibition of endocannabinoid activity in the cell expressing CB₁R/A2aRheteromer and in the control cell in the presence of the compound, andc) selecting the compound which preferentially inhibits endocannabinoidactivity in the cell expressing the CB₁R/A2aR heteromer as compared tothe control cell, as the candidate compound useful for the treatment ofthe liver disease.

In one embodiment, the inhibition of endocannabinoid activity isdetermined by measuring adenosine-mediated ERK1/2 phosphorylation.Preferably, the inhibition of endocannabinoid activity in the cellexpressing the CB₁R/A2aR heteromer is at least about 5-fold greater thanthe inhibition of endocannabinoid activity in the control cell. Inanother preferred embodiment, the cell expressing the CB₁R/A2aRheteromer, CB₁R alone, or A2aR alone is HEK293 or CHO cell.

In another preferred embodiment, a method of screening for a compounduseful for treating a liver disease, the method comprises a) contactingthe compound with a cell expressing a type I cannabinoid receptor(CB₁R)/angiotensin II receptor (AT1R) heteromer, and with a control cellexpressing CB₁R alone or AT1R alone; b) measuring decrease inintracellular cAMP levels in the cell expressing CB₁R/AT1R heteromer andin the control cell in the presence of the compound, and c) selectingthe compound which causes a greater decrease in intracellular cAMPlevels in the cell expressing the CB₁R/AT1R heteromer as compared to thecontrol cell, as the candidate compound useful for the treatment of theliver disease. In one embodiment, the intracellular cAMP levels aremeasured using a cAMP detection kit. Preferably, the decrease inintracellular cAMP levels in the cell expressing the CB₁R/AT1R heteromeris about at least 5 fold greater than the decrease in intracellular cAMPlevels in the control cell.

In another preferred embodiment, a method of screening for adiacylglycerol lipase (DAGL) inhibitor, the method comprises a)contacting a compound with a cell expressing a type I cannabinoidreceptor (CB₁R) and a G-protein coupled receptor (GPCR), each receptorhaving a respective ligand, wherein the receptors are only capable ofinducing ERK1/2 phosphorylation when the CB₁R and the GPCR areco-stimulated by a ligand, b) measuring changes in ERK1/2phosphorylation in the presence of the compound, and c) selecting thecompound which causes a decrease in ERK1/2 phosphorylation as the DAGLinhibitor.

In one embodiment, the GPCR is AT1R or A2aR.

In another preferred embodiment, the compound to be screened ispre-incubated with the CB1R and GPCR ligands and added to the cells.Preferably, the screen is a high-throughput screen.

In another preferred embodiment, a method of measuring selective bindingof a compound to CB1R/AT1R or CB1R/A2aR heteromer, the method comprisesa) providing a cell expressing CB1R/AT1R or CB1R/A2aR heteromer and acontrol cell expressing only CB1R, only AT1R, or only A2aR; b)contacting the cell expressing CB1R/AT1R or CB1R/A2aR heteromer and thecontrol cell with Angiotensin II or A2aR agonist or the compound; c)adding a radioactive labeled Angiotensin II or A2aR agonist; and, d)measuring receptor-bound radioactivity. In one embodiment, the detectionof the radiolabel is determinative of selective binding.

In another preferred embodiment, an isolated monoclonal antibodyspecifically binds to a type I cannabinoid receptor (CB₁R)/adenosine 2areceptor (A2aR) heteromer.

These and other aspects of the present invention will be apparent tothose of ordinary skill in the art in light of the presentspecification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H: Isolation of CB₁R/AT1R and CB₁R/A2aR complexes in activatedhepatic stellate cells (HSCs) and fibrotic livers. Lysates weresubjected to SDS-PAGE and immunoblotting. The levels of Ang II-mediatedpERK normalized to total ERK were measured by Western blot. Data areexpressed as the mean±SD (n=3-4).

FIG. 1A. Comparison of the expression levels of CB₁R, AT1R, and α-SMA.Western blot in HSCs from control (cHSC) and ethanol (eHSCs) treatedrats.

FIG. 1B. Association of AT1R and CB₁R in eHSCs. Lysates from cHSCs andeHSCs were subjected to immunoprecipitation using an anti-AT1Rantibody/protein A/C agarose complex, and to Western blotting. CB₁R isdetected in the AT1R-immunoprecipitate from eHSCs.

FIG. 1C. Comparison of the expression levels of A2aR by Western blot inHSCs from control (cHSC) and ethanol (eHSCs) treated rats.

FIG. 1D. Association of A2aR and CB₁R in eHSCs. Lysates from cHSCs andeHSCs were subjected to immunoprecipitation using an anti-A2aRantibody/protein A/C agarose complex, and to Western blotting. CB₁R isdetected in the A2aR immunoprecipitate from eHSCs.

FIG. 1E. Comparison of the expression levels of CB₁R, AT1R, A2aR andPDGFRβ by Western blot in livers from vehicle and CCl₄ treated mice.

FIG. 1F. Association of AT1R or A2aR with CB₁R in mouse fibrotic livers.Lysates from the liver of vehicle or CCl₄ treated mice were subjected toimmunoprecipitation using an anti-AT1R or an anti-A2aR antibody/proteinA/C agarose complex, and to Western blotting. CB₁R is detected in theAT1R and A2aR immunoprecipitates from livers from CCl₄ treated mice.

FIG. 1G. Comparison of the expression levels of CB₁R, AT1R, A2aR, PDGFRβand α-SMA by Western blot in livers from control and cirrhotic humanpatients.

FIG. 1H. Association of A2aR and CB₁R cirrhotic human livers. Lysatesfrom control and cirrhotic human livers were subjected toimmunoprecipitation using an anti-A2aR antibody/protein A/C agarosecomplex, and to Western blotting. CB₁R is detected in the A2aRimmunoprecipitate from eHSCs, and there is enhanced association inlivers from cirrhotic patients as compared to control patients.

FIGS. 2A-2C: CB₁R activity controls AT1R and A2aR signaling in activatedHSCs.

FIG. 2A. CB₁R antagonist prevents Ang II-mediated pERK in eHSCs. cHSCsand eHSCs were stimulated with Ang II in the absence or presence ofSR141716 (SR).

FIG. 2B. Blocking the production of endocannabinoids reduces AngII-mediated pERK. cHSCs and eHSCs pretreated with THL for 10 or 30minutes were stimulated with Ang II.

FIG. 2C. CB₁R antagonist prevents CGS21680 mediated pERK in eHSCs. eHSCswere stimulated with CGS21680 (CGS; specific adenosine A2A subtypereceptor (A2aR) agonist) in the absence or presence of SR. Lysates weresubjected to SDS-PAGE and immunoblotting. The levels of Ang II- orCGS-mediated pERK normalized to total ERK are indicated. Western blotdata are expressed as the mean±SD (n=3-4). *p<0.05; **p<0.01, Student'st test.

FIGS. 3A-3E. Characterization of the CB₁R-AT1R complex.

FIG. 3A. CB₁R activation regulates Ang II-mediated increase in pERK inHEK293 cells. HEK293 transfected with AT1R alone or together with CB₁Rplasmids were stimulated with 10 nM Ang II for 3 minutes, in the absenceor presence of SR141716 (SR; 1 μM); or THL (1 μM; 1 hour pre-treatment).ERK phosphorylation was assessed by Western blot using antibodies topERK and ERK. Data represent mean±SEM (n=4).

FIG. 3B. Functional interaction between CB₁R and AT1R. The levels of AngII-mediated pERK normalized to total ERK are summarized under varioustreatments, after 10 nM Ang II stimulation for 3 minutes.

FIGS. 3C and 3D. Endocannabinoid-mediated CB₁R basal activity allows AngII-mediated pERK. PhosphoERK levels after Ang II stimulation wereexamined in Neuro2A-AT1R cells, after treatment with the diacylglycerollipase inhibitor THL alone or together with Hu210 (0.1 nM) (FIG. 3C); orwith increasing concentrations of 2-AG (FIG. 3F). Lysates were subjectedto SDS-PAGE and immunoblotting. Data are expressed as the mean±SD(n=3-4).

FIG. 3E. Association with CB₁R affects AT1R G protein coupling.PhosphoERK (pERK) levels after Ang II stimulation were examined inNeuro2A-AT1R cells, after incubation with pertussis toxin (PTX) withoutor with RNAi-mediated CB₁R downregulation; after transfection with a Gαqdominant negative construct (DN Gαq) without or with RNAi-mediated CB₁Rdownregulation.

FIGS. 4A-4C. CB₁R controls the profibrogenic activity of AT1R and A2aR.For analysis of gene expression, after reverse transcription, the numberof copies of mRNA for the indicated transcripts were determined byreal-time PCR. Data were normalized to GAPDH mRNA and are expressed asthe mean±SD (n=3 in quadruplicate).

FIG. 4A. CB₁R antagonist prevents Ang II-mediated fibrogenic response ineHSC. eHSCs were stimulated with Ang II in the absence or presence ofSR141716 (SR) for 4 hours before the RNA was harvested.

FIG. 4B. CB₁R antagonist prevents CGS21680 (CGS)-mediated fibrogenicresponse in eHSC. eHSCs were stimulated with CGS in the absence orpresence of SR for 6 hours before the RNA was harvested.

FIG. 4C. CB₁R antagonist prevents CGS-mediated fibrogenic response inmouse fibrotic liver. CCl₄- or vehicle-treated mice were injectedintraperitoneally (i.p.) with vehicle, CGS, or CGS+AM 251 (AM, CB₁Rantagonist). After 6 hours, the mice were sacrificed, their liverquickly removed and the RNA harvested.

FIG. 5. Phospho-ERK expression following treatment with THL and 2-AG.Neuro2A-AT1 were pre-treated with THL (2 μM, 3 h) in serum free mediaand stimulated with increasing concentrations of 2-AG in the presence ofAng 1 for 3 minutes. Cells were lysed, and cell lysates were probed forthe levels of pERK1/2 and ERK 1/2.

FIG. 6. Effect of THL on Ang II-mediated ERK phosphorylation inNeuro2A-AT1 cells. Cells were pretreated with THL (1 μM) for two hours,then they were stimulated with 10 nM Ang II in the presence or absenceof 2-AG (100 nM), for 3 minutes. ERK phosphorylation was assessed byWestern blot using antibodies to pERK and ERK. N=3-5.

FIG. 7. High through-put screening assay. 24 h after plating, the cellswere starved for 2 hours, then incubated with vehicle (DMSO) or with THL(1 μM) for 2 hours, then stimulated with Ang II (10 nM) for 3 minutes.The cells were processed for infrared detection of pERK.

FIG. 8A-8G. Characterization of CB1R-AT1R Antibody Binding Specificityand Biological Activity.

FIG. 8A. Detection of AT1R-CB1R heteromers with heteromer-selectivemonoclonal antibodies. Receptor abundance was determined in Neuro2A,Neuro2A-AT1R and Neuro2A-AT1R cells where CB1R was downregulated by RNAi(Neuro2A-AT1R siCB1R) with a monoclonal antibody to AT1R-CB1R, orpolyclonal antibodies to AT1R or to CB1R by ELISA. Results are means±SEM(n=3 experiments). ***, p<0.001; n.s., non-significant, vs Neuro2A-AT1R.

FIG. 8B. HEK293 cells were transfected with plasmids coding for CB1R andAT1R, alone or in combination. Receptor abundance was determined withthe monoclonal antibody to AT1R-CB1R, or polyclonal antibodies to AT1Ror to CB1R by ELISA. Results are means±SEM (n=3).

FIG. 8C. HEK293 cells transfected with plasmids coding for the indicatedGPCRs were incubated with the antibody to AT1R-CB1R and subjected toELISA. Results are means±SEM (n=3).

FIG. 8D. HEK293 cells were transfected with different ratios of theplasmids coding for Myc-CB1R and Flag-AT1R, as indicated. The cells wereincubated with the indicated antibodies and subjected to ELISA. Resultsare means±SEM (n=3).

FIG. 8E. Detection of AT1R-CB1R heteromers with heteromer-selectivemonoclonal antibodies in the activated hepatic stellate cells (eHSCs).Receptor abundance was determined in cHSCs and eHSCs with a monoclonalantibody to AT1R-CB1R by ELISA. Results represent the means±SEM obtainedwith antibodies from 7 different hybridoma clones. ***, p<0.001.

FIG. 8F. CB1R-AT1R mediated activity can be selectively blocked byheteromer specific antibody. Neuro-2A cells (40,000 cells/well)endogenously expressing CB1R and transfected with AT1R were preincubatedwithout or with heteromer antibodies followed by treatment with 1 μM AngII (AT1R agonist) in the absence or presence of 10 nM AM (CB1Rantagonist) and pERK levels were determined by Western blotting.

FIG. 8G. Inhibition of AT1R-CB1R signaling by the AT1R-CB1R heteromerantibody. Neuro2A-AT1R and Neuro2A-AT1R cells where CB1R wasdownregulated by RNAi (Neuro2A-AT1R/siCB1R) were incubated withincreasing concentrations of the monoclonal anti-AT1R-CB1R antibody(hydridoma supernatant, +, 1:20 v/v; ++, 1:10 v/v; +++, 1:5 v/v; ++++,2:5 v/v) for 30 minutes, and then were stimulated with 10 nM Ang II for3 minutes. Cell lysates and media were subjected to Western blottinganalysis using antibodies to pERK and ERK (1:1,000) (lysate) andanti-mouse IgG (media). Imaging and quantification was carried out usingthe Odyssey Imaging system (Li-Core Biosciences). Results are means±SEM(n=4 experiments). ***, p<0.001; n.s., non-significant, vs thecorresponding Ang II treatment.

FIGS. 9A-9D: Characterization of Ang II-mediated increased in pERK inNeuro2A-AT1R cells. Data represent mean±SEM (n=2-5).

FIG. 9A. Association of AT1R and CB₁R in Neuro2A-AT1R. Lysates fromNeuro2A and Neuro2A-AT1R were subjected to immunoprecipitation using aprotein A-agarose coupled-anti-CB₁R antibody, and to Western blotting.AT1R is detected in the CB₁R immunoprecipitate from Neuro2A-AT1R.

FIG. 9B. Time course of Ang II-mediated increase in pERK. Neuro2A-AT1Rcells were stimulated with 10 nM Ang II in the absence or presence ofthe AT1R specific antagonist Losartan (1 μM) for the indicated times.

FIG. 9C. Dose response of ERK phosphorylation to Ang II treatment inNeuro2A-AT1R. Cells were stimulated with increasing concentration of AngII for 3 minutes. It is to be noted that concentrations of Ang IIsuperior to 10 nM lead to a decrease in pERK levels, demonstrating adesensitization of the pathway leading to ERK phosphorylation at highconcentrations.

FIG. 9D. Influence of CB₁R on Ang II-mediated increase in pERK.Neuro2A-AT1R transfected or not with a siRNA to CB₁R, or Neuro2A (wildtype) cells were stimulated with 10 nM Ang II for the indicated times.ERK phosphorylation was assessed by Western blot using antibodies topERK and ERK.

FIGS. 10A-10C: CB₁R regulates Ang II-mediated increase in pERK. ERKphosphorylation was assessed by Western blot using antibodies to pERKand ERK. Data represent mean±SEM (n=3-5).

FIG. 10A. Dose response of ERK phosphorylation to Ang II treatment inNeuro2A-AT1R transfected or not with a siRNA to CB₁R.

FIG. 10B. Dose response of ERK phosphorylation to Ang II treatment inthe presence of CB₁R antagonist (SR141716; 1 μM).

FIG. 10C. Dose response of ERK phosphorylation to Ang II treatment inthe presence of a non-signaling dose of the CB₁R agonist (Hu210; 0.1nM). Cells were stimulated with increasing concentration of Ang II.

FIGS. 11A-11D: CB₁R switches AT1R coupling to Gαi. Cells were stimulatedwith increasing concentration of Ang II. ERK phosphorylation wasassessed by Western blot using antibodies to pERK and ERK. Datarepresent mean±SEM (n=3-5).

FIG. 11A. Dose response of ERK phosphorylation to Ang II treatment inNeuro2A-AT1R transfected or not with a siRNA to CB₁R and treated or notwith Pertussis Toxin (PTX, 15 nM, 16 hours).

FIG. 11B. Dose response of ERK phosphorylation to Ang II treatment inNeuro2A-AT1R transfected or not with a dominant negative Gαq constructalone or together with a siRNA to CB₁R.

FIG. 11C. Dose response of ERK phosphorylation to Ang II treatment inNeuro2A-AT1R treated with vehicle or forskolin (1 μM, 30 min).

FIG. 11D. Dose response of ERK phosphorylation to Ang II treatment inNeuro2A-AT1R transfected or not with a siRNA to βarrestin2.

FIGS. 12A-12B. Preparation of the cHSC and eHSC isolated from alcoholfed rats. Rats (300 g female Sprague-Dawley, N=10/group) were fed thecontrol or ethanol Lieber-DeCarli diets for 8 months.

FIG. 12A. Ultrastructural analysis depicting micro- and macrovesicularsteatosis, vacuolization, and electron dense mitochondria in livers fromethanol fed rats (right panel) compared to livers from control rats(left panel) (magnification=2500×).

FIG. 12B. Light micrographs from HSC isolated from control andethanol-fed rats.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to compounds and compositions thereof,useful for the treatment of liver disease and methods of treating liverdisease. The present invention also relates to methods of screening forcompounds useful for the treatment of liver disease. The presentinvention also relates to methods of screening for diacylglycerol lipaseinhibitors.

The present invention demonstrates that there is enhanced,fibrosis-specific association of CB₁R with angiotensin II receptor(AT1R) and with adenosine A2A receptors. These complexes have a uniquetissue localization and expression during a pathological state (namely,liver fibrosis). Therefore, drugs targeting these receptor complexeswill have enhanced selectivity (both spatial and temporal) withpotentially fewer side effects and undesired properties than drugstargeting either of the heteromer components individually, out of thecontext of the heteromer (e.g., as monomers or homodimers). This findinghas resulted in the identification of novel compounds and methods oftreating liver diseases, such as cirrhosis and fibrosis, and novelmethods of identifying compounds useful for the treatment of liverdisease.

The endocannabinoid 2-AG is highly upregulated during chronic liverdiseases and is implicated in the pathogenesis of non-alcoholic fattyliver disease, progression of fibrosis to cirrhosis and the developmentof the cardiovascular abnormalities of cirrhosis, such as thehyperdynamic circulatory syndrome and cirrhotic cardiomiopathy. Thedesign or identification of compounds that inhibits 2-AG activity isimportant for the treatment of such conditions. The present inventionalso provides for a receptor complex system in which ERK1/2phosphorylation can be controlled in a concentration-dependent manner by2-AG.

Several aspects of the invention are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. The present invention is not limited by the illustratedordering of acts or events, as some acts may occur in different ordersand/or concurrently with other acts or events. Furthermore, not allillustrated acts or events are required to implement a methodology inaccordance with the present invention.

All genes, gene names, and gene products disclosed herein are intendedto correspond to homologs from any species for which the compositionsand methods disclosed herein are applicable. Thus, the terms include,but are not limited to genes and gene products from humans and mice. Itis understood that when a gene or gene product from a particular speciesis disclosed, this disclosure is intended to be exemplary only, and isnot to be interpreted as a limitation unless the context in which itappears clearly indicates. Thus, for example, for the genes disclosedherein, which in some embodiments relate to mammalian nucleic acid andamino acid sequences are intended to encompass homologous and/ororthologous genes and gene products from other animals including, butnot limited to other mammals, fish, amphibians, reptiles, and birds. Inpreferred embodiments, the genes or nucleic acid sequences are human.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art andwill not be interpreted in an idealized or overly formal sense unlessexpressly so defined herein.

Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

As used herein, the term “about” or “approximately” usually means withinan acceptable error range for the type of value and method ofmeasurement. For example, it can mean within 20%, more preferably within10%, and most preferably still within 5% of a given value or range.Alternatively, especially in biological systems, the term “about” meanswithin about a log (i.e., an order of magnitude) preferably within afactor of two of a given value.

A “heteromer” is a protein complex formed from two or more differenttypes of subunits or components. A GPCR heteromer is a complex formedfrom two or more different GPCR molecules (protomers). In a specificembodiment, a heteromer is a heterodimer. In preferred embodiments, aheteromer comprises: CB₁R complexed with angiotensin II receptor (AT1R)(CB₁R-AT1R) or with adenosine A2A receptors (CB₁R-A2aR).

The term “inhibiting” a heteromer is meant to include inhibiting atleast one function or formation of the heteromer. For example,inhibiting the activity of a CB₁R-AT1R heteromer, includes, amongothers, inhibiting formation of the complex, inhibiting ERKphosphorylation, and the like. Assays to measure the differentactivities of heteromers and their inhibition are described in detail inthe examples section which follows.

“Endocannabinoid activity” refers to any one or more in vivo functionsof the molecules involved in endocannabinoid system, for example,stimulation of CB R resulting in activation of the ERK1/2 pathway. Theendocannabinoid system refers to a group of neuromodulatory lipids andtheir receptors that are involved in a variety of physiologicalprocesses including appetite, pain-sensation, mood, and memory. It isnamed for endocannabinoids, the endogenous lipids that bind cannabinoidreceptors (the same receptors that mediate the psychoactive effects ofcannabis). Broadly speaking, the endocannabinoid system refers to: thecannabinoid receptors CB1 and CB2, two G protein-coupled receptorsprimarily located in the central nervous system and periphery,respectively. The endogenous arachidonate-based lipids, anandamide(N-arachidonoylethanolamine (AEA)) and 2-arachidonoylglycerol (2-AG),collectively termed the “endocannabinoids”, that are ligands for thecannabinoid receptors. Enzymes synthesize and degrade theendocannabinoids anandamide and 2-AG. Unlike neurotransmitters,endogenous cannabinoids are not stored in vesicles after synthesis, butare synthesized on demand. In preferred embodiments, the compounds ofthe invention, modulate or inhibit endocannabinoid activity for thetreatment of liver fibrosis, liver diseases, obesity, metabolicsyndrome, pain, affective and neurodegenerative disorders, inflammation.

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.The term “antibody”, is inclusive of all species, including human andhumanized antibodies. The antigenic target, for example, CB₁R-AT1Rheteromers, CB₁R-A2aR heteromers, can be also be from any species.Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H-CH1) by a disulfide bond. TheF(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into anFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term “antibody”, asused herein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Embodiments include single chainantibodies, single chain Fv (scFv) antibodies in which a variable heavyand a variable light chain are joined together (directly or through apeptide linker) to form a continuous polypeptide.

An “antigen-binding site” or “binding portion” refers to the part of animmunoglobulin molecule that participates in antigen binding. The phrase“specifically binds to” when referring to an antibody refers to abinding reaction which is determinative of the presence of the proteinor peptide in the presence of a heterogeneous population of proteins andother biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein and do not bind in asignificant amount to other proteins present in the sample. Specificbinding to a protein under such conditions may require an antibody thatis selected for its specificity for a particular protein. For example,antibodies specific for CB₁R-AT1R heteromers or CB₁R-A2aR heteromers canbe raised to the CB₁R-AT1R heteromers or CB₁R-A2aR heteromers peptidesthat specifically bind to each heteromer protein or peptides thereof,and not to other proteins present in a tissue sample. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity. As used herein, the term“binding affinity” refers to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength or binding affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (Kd) of the interaction, wherein a smaller Kdrepresents a greater affinity. Binding properties of selectedpolypeptides can be quantified using methods well known in the art. Onesuch method entails measuring the rates of antigen-binding site/antigencomplex formation and dissociation, wherein those rates depend on theconcentrations of the complex partners, the affinity of the interaction,and on geometric parameters that equally influence the rate in bothdirections. Thus, both the “on rate constant” (K_(on)) and the “off rateconstant” (K_(off)) can be determined by calculation of theconcentrations and the actual rates of association and dissociation. Theratio of K_(off)/K_(on) enables cancellation of all parameters notrelated to affinity and is thus equal to the dissociation constant Kd.See, generally, Davies et al. Ann. Rev. Biochem., 59: 439-473 (1990). Inone embodiment, the invention includes antibodies that bind to theheteromers with very high affinities (Kd). For example a human, rabbit,mouse, chimeric or humanized antibody that is capable of bindingCB1R-AT1R heteromers or CB1R-A2aR heteromers with a Kd less than, butnot limited to, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰ or 10⁻¹¹ M, or anyrange or value therein. Thus, an antibody with a “high-affinity” is anantibody having a Kd, or dissociation constant, in the nanomolar (nM)range or better. A Kd in the “nanomolar range or better” may be denotedby X nM, where X is a number less than about 10. Affinity and/or aviditymeasurements can be measured by ligand binding assays, KinExA™ and/orBIACORE™.

The antibody specificities are also meant to encompass any variants,mutants, derivatives, alleles, isoforms, orthologs, fragments ofCB₁R-AT1R heteromers or CB₁R-A2aR heteromers. Thus, an antibody orantigen-binding portion thereof of the invention specifically binds tothe heteromer of CB₁R/AT1R or CB₁R/A2aR, rather than to monomers orhomodimers of the components of the complex (i.e., the antibody orantigen-binding portion thereof specifically recognizes an epitope thatis only available when the heteromer complex is formed). The increase inbinding of the antibody to the heteromer complex compared to binding tomonomers or homodimers of CB1R or AT1R is at least about 5-fold. Theantibodies preferably bind to the heteromers rather than the monomers.

The terms “monoclonal antibody” and “monoclonal antibody composition” asused herein, refer to a preparation consisting of a single species ofantibody molecules with a single binding specificity and affinity for aparticular epitope within the antigen. A monoclonal antibody refers to ahomogeneous antibody population wherein the monoclonal antibody iscomprised of amino acids (naturally occurring and non-naturallyoccurring) that are involved in the selective binding of an antigen.Monoclonal antibodies are highly specific, being directed against asingle antigenic site. The term “monoclonal antibody” encompasses notonly intact monoclonal antibodies and full-length monoclonal antibodies,but also fragments thereof, mutants thereof, fusion proteins comprisingan antibody portion, humanized monoclonal antibodies, chimericmonoclonal antibodies, and any other modified configuration of theimmunoglobulin molecule that comprises an antigen recognition site ofthe required specificity and the ability to bind to an antigen. It isnot intended to be limited as regards to the source of the antibody orthe manner in which it is made (e.g., by hybridoma, phage selection,recombinant expression, transgenic animals, etc.). Additionally,techniques for the production and isolation of monoclonal antibodies andantibody fragments are known in the art, and are generally described,among other places, in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988, and in Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, London, 1986.

The term “human monoclonal antibody” or “humanized antibodies” refers tomonoclonal antibodies that have variable and constant regions (ifpresent) derived from human germline immunoglobulin sequences.“Humanized” antibodies refer to molecules having an antigen binding sitethat is substantially derived from an immunoglobulin from a non-humanspecies and the remaining immunoglobulin structure of the molecule basedupon the structure and/or sequence of a human immunoglobulin. Theantigen binding site may comprise either complete variable domains fusedonto constant domains or only the complementarity determining regions(CDRs) grafted onto appropriate framework regions in the variabledomains. Antigen binding sites may be wild type or modified by one ormore amino acid substitutions, e.g., modified to resemble humanimmunoglobulin more closely. Some forms of humanized antibodies preserveall CDR sequences (for example, a humanized mouse antibody whichcontains all six CDRs from the mouse antibodies). Other forms ofhumanized antibodies have one or more CDRs (one, two, three, four, five,or six) which are altered with respect to the original antibody. Humanmonoclonal antibodies can be produced by standard methodologies,including though the preparation of an appropriate hybridoma.

The term “epitope” includes any molecular (e.g., protein) determinantcapable of being specifically bound by an immunoglobulin molecule orT-cell receptor. Epitopic determinants usually consist of chemicallyactive surface groupings of molecules, such as amino acids or sugar sidechains, and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.

The term “agonist” or “agonist-like” as used herein means that upon thebinding of an anti-CB₁R/GPCR heteromer antibody of the present invention(e.g., anti-CB₁R/AT1R or CB₁R/A2aR heteromer antibody) orantigen-binding portion thereof, to the CB₁R/AT1R or CB₁R/A2aR heteromer(“the antibody target”), at least one biological effect typicallyobserved when a natural ligand of the target heteromer binds to thereceptor complex in vivo is induced. For example, an anti-CB₁R/AT1Rheteroimer antibody having an agonist-like effect will induce at leastone biological effect typically observed following stimulation of theheteromer by 2-AG, a natural ligand of the receptor complex. Such aneffect can be, for example, a decrease in the production ofintracellular cAMP.

The term “antagonist” or “antagonist-like” as used herein means thatupon the binding of an anti-CB₁R/GPCR heteromer antibody of the presentinvention (e.g., anti-CB₁R/AT1R or CB₁R/A2aR heteromer antibody) orantigen-binding portion thereof, to the antibody target, at least onebiological effect typically observed when a natural ligand of the targetheteromer binds to the receptor complex in vivo is inhibited partiallyor fully. For example, an anti-CB₁R/AT1R heteromer antibody having anantagonist-like effect will reduce or inhibit at least one biologicaleffect typically observed following stimulation of the heteromer by2-AG, a natural ligand of the receptor complex. Such an effect can be,for example, a decrease the production of intracellular cAMP normallyinduced by an agonist of the subject heteromer complex.

As used herein, the terms “isolated” and “isolating” mean that thereferenced material is removed from its native environment, e.g., acell. Thus, an isolated biological material can be free of some or allcellular components, i.e., components of the cells in which the nativematerial naturally occurs (e.g., a cytoplasmic or membrane component).

“Detectable moiety” or a “label” refers to a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, ³⁵S, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin-streptavidin, dioxigenin, haptens and proteins for which antiseraor monoclonal antibodies are available, or nucleic acid molecules with asequence complementary to a target. The detectable moiety oftengenerates a measurable signal, such as a radioactive, chromogenic, orfluorescent signal, that can be used to quantify the amount of bounddetectable moiety in a sample. Quantitation of the signal is achievedby, e.g., scintillation counting, densitometry, or flow cytometry.

The term “liver disease” includes diseases and conditions of the liverincluding liver cirrhosis, alcoholic and non-alcoholic fibrosis as wellas to liver disease or changes associated with obesity, diabetes andmetabolic syndrome. Other examples of liver diseases include: hepatitis,fatty liver, toxic liver failure, hepatic cirrhosis, diabetes-associatedliver disease, liver steatosis, liver fibrosis, liver cirrhosis, chronichepatitis and the like.

As used herein, the term “test substance” or “candidate therapeuticagent” or “agent” or “compound” are used interchangeably herein, and theterms are meant to encompass any molecule, chemical entity, composition,drug, therapeutic agent, chemotherapeutic agent, or biological agentcapable of preventing, ameliorating, or treating a disease or othermedical condition. The term includes small molecule compounds, antisensereagents, siRNA reagents, antibodies, enzymes, peptides organic orinorganic molecules, natural or synthetic compounds and the like. A testsubstance or agent can be assayed in accordance with the methods of theinvention at any stage during clinical trials, during pre-trial testing,or following FDA-approval.

As used herein, “modulation” means either an increase (stimulation) or adecrease (inhibition) in the expression, in vivo amounts of a targetmolecule, e.g. CB₁R-AT1R heteromers, CB₁R-A2aR heteromers. This includesany amounts in vivo, functions and the like as compared to normalcontrols. The term includes, for example, increased, enhanced,increased, agonized, promoted, decreased, reduced, suppressed blocked,or antagonized. Modulation can increase activity or amounts more than1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, etc., over baselinevalues. Modulation can also decrease its activity or amounts belowbaseline values.

As used herein the phrase “diagnostic” means identifying the presence ornature of a pathologic condition. Diagnostic methods differ in theirsensitivity and specificity. The “sensitivity” of a diagnostic assay isthe percentage of diseased individuals who test positive (percent of“true positives”). Diseased individuals not detected by the assay are“false negatives.” Subjects who are not diseased and who test negativein the assay are termed “true negatives.” The “specificity” of adiagnostic assay is 1 minus the false positive rate, where the “falsepositive” rate is defined as the proportion of those without the diseasewho test positive. While a particular diagnostic method may not providea definitive diagnosis of a condition, it suffices if the methodprovides a positive indication that aids in diagnosis.

As used herein the phrase “diagnosing” refers to classifying a diseaseor a symptom, determining a severity of the disease, monitoring diseaseprogression, forecasting an outcome of a disease and/or prospects ofrecovery. The term “detecting” may also optionally encompass any of theabove. Diagnosis of a disease according to the present invention can beeffected by determining a level of a polynucleotide or a polypeptide ofthe present invention in a biological sample obtained from the subject,wherein the level determined can be correlated with predisposition to,or presence or absence of the disease. It should be noted that a“biological sample obtained from the subject” may also optionallycomprise a sample that has not been physically removed from the subject.

The term “sample” is meant to be interpreted in its broadest sense. A“sample” refers to a biological sample, such as, for example; one ormore cells, tissues, or fluids (including, without limitation, plasma,serum, whole blood, cerebrospinal fluid, lymph, tears, urine, saliva,milk, pus, and tissue exudates and secretions) isolated from anindividual or from cell culture constituents, as well as samplesobtained from, for example, a laboratory procedure. A biological samplemay comprise chromosomes isolated from cells (e.g., a spread ofmetaphase chromosomes), organelles or membranes isolated from cells,whole cells or tissues, nucleic acid such as genomic DNA in solution orbound to a solid support such as for Southern analysis, RNA in solutionor bound to a solid support such as for Northern analysis, cDNA insolution or bound to a solid support, oligonucleotides in solution orbound to a solid support, polypeptides or peptides in solution or boundto a solid support, a tissue, a tissue print and the like. Numerous wellknown tissue or fluid collection methods can be utilized to collect thebiological sample from the subject in order to determine the level ofDNA, RNA and/or polypeptide of the variant of interest in the subject.Examples include, but are not limited to, fine needle biopsy, needlebiopsy, core needle biopsy and surgical biopsy (e.g., brain biopsy), andlavage. Regardless of the procedure employed, once a biopsy/sample isobtained the level of the variant can be determined and a diagnosis canthus be made.

“Treating” or “treatment” covers the treatment of a disease-state in amammal, and includes: (a) preventing the disease-state from occurring ina mammal, in particular, when such mammal is predisposed to thedisease-state but has not yet been diagnosed as having it; (b)inhibiting the disease-state, e.g., arresting it development; and/or (c)relieving the disease-state, e.g., causing regression of the diseasestate until a desired endpoint is reached. Treating also includes theamelioration of a symptom of a disease (e.g., lessen the pain ordiscomfort), wherein such amelioration may or may not be directlyaffecting the disease (e.g., cause, transmission, expression, etc.).

The phrase “therapeutically effective amount” means an amount of acompound or a combination of compounds that ameliorates, attenuates oreliminates one or more of the symptoms of a particular disease orcondition or prevents, modifies, or delays the onset of one or more ofthe symptoms of a particular disease or condition. Thus, atherapeutically effective amount/dose refers to the amount/dose of apharmaceutical composition of the invention that is suitable fortreating a patient or subject having, for example, a liver disease suchas liver fibrosis or cirrhosis. In certain embodiments of the inventionthe patient or subject may be a mammal. In certain embodiments, themammal may be a human.

As used herein, the term “animal” or “patient” is meant to include, forexample, humans; domestic, veterinary or farm animals such as, forexample, sheep, elks, deer, mule deer, minks, mammals, monkeys, horses,cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken, reptiles,fish, insects and arachnids.

“Mammal” covers warm blooded mammals that are typically under medicalcare (e.g., humans and domesticated animals). Examples include feline,canine, equine, bovine, and human, as well as just human.

The term “ED₅₀” means the dose of a drug that produces 50% of itsmaximum response or effect.

The term “IC₅₀” means the concentration of a drug which inhibits anactivity or property by 50%, e.g., by reducing the frequency of acondition, such as cell death, by 50%, by reducing binding of acompetitor peptide to a protein by 50% or by reducing the level of anactivity by 50%.

The term “LD₅₀” means the dose of a drug that is lethal in 50% of testsubjects.

“Composition” indicates a combination of multiple substances into anaggregate mixture.

Screening Assays

In the present invention, methods of screening for a compound that isuseful for the treatment of liver disease are provided. In certainembodiments, such methods are useful for identifying a compound thatspecifically binds to the CB₁R/adenosine 2a receptor (A2aR) heteromer.These methods include the steps of: a) mixing the compound with theCB₁R/A2aR heteromer, CB₁R monomers, CB₁R homodimers, A2aR monomers, andA2aR homodimers, b) measuring the amount of compound bound to theCB₁R/A2aR heteroer, CB₁R monomers, CB₁R homodimers, A2aR monomers, andA2aR homodimers, and c) selecting as a compound that specifically bindsto the CB₁R/A2aR heteromer as compared to binding to control receptorssuch as, for example, CB₁R monomers, CB₁R homodimers, A2aR monomers, orA2aR homodimers. Preferably, the compound inhibits the activity of theheteromers. The activity can include heteromers complex formation,phosphorylation of ERK etc.

In certain aspects of the present invention, the methods for screening acompound that is useful for the treatment of liver disease may also becarried out using the CB₁R/angiotensin II receptor (AT1R) heteromer.Similarly, such methods are useful for identifying a compound thatspecifically binds to the CB₁R/AT1R heteromer as compared to eachmonomer subunit. These methods include the steps of: a) mixing thecompound with the CB₁R/AT1R heteromer, CB₁R monomers, CB₁R homodimers,AT1R monomers, and AT1R homodimers, b) measuring the amount or ofcompound bound to the CB₁R/AT1R heteromer, CB₁R monomers, CB₁Rhomodimers, AT1R monomers, and AT1R homodimers, and c) selecting as acompound that specifically binds to the CB₁R/AT1R heteromer and inhibitsthe activity of the heteromers as compared to binding to controlreceptors such as, for example, CB₁R monomers, CB₁R homodimers, AT1Rmonomers, or AT1R homodimers.

In certain embodiments of the present invention, specific binding of ascreening compound of the invention to the CB₁R/AT1R and/or CB₁R/A2aRheteromers is measured by a classical ligand binding assay. For example,a ligand binding assay may be carried out in cells, such as, forexample, CHO, HEK293, COS cells, and the like, expressing only CB₁R,only AT1R, only A2aR, or co-expressing CB₁R/AT1R or CB₁R/A2aR. Adetailed protocol for a ligand binding assay is provided in the Examplessection which follows.

Specific binding of a compound to heteromers of the invention (CB₁R/A2aRor CB₁R/AT1R) i.e., a composition exhibiting a higher affinity ascompared to a control, is preferably at least about 5 fold greater, morepreferably at least about 10 fold greater, and most preferably at leastabout 20 fold greater than binding to the homodimers or monomers of themembers comprising the heteromer to which the composition binds (e.g.,of A2aR, AT1R or CB₁R). In another preferred embodiment, a compound mayinhibit the function or activity of any of the heteromers by modulatingone or more activities, such as for example, signaling, phosphorylationof ERK, endocannabinoid activity, profribrogenic activity, binding ofligands to the heteromer, and the like.

The term “compound” is meant to cover any molecule or molecules thatmodulate the function or activities in vivo or in vitro, of theCB₁R/AT1R and/or CB₁R/A2aR heteromers, including the inhibition ofheteromer formation. The compounds are specific for the heteromers ascompared to the monomer subunits or control receptors, e.g. CB₁Rmonomers, CB₁R homodimers, AT1R monomers, AT1R homodimers, A2aRmonomers, or A2aR homodimers. Examples of compounds comprise: aptamers,antibodies, small molecules, nucleic acids, proteins, peptides,polypeptides, peptidomimetics, peptoids enzymes, organic or inorganicmolecules, synthetic molecules, natural molecules, or combinationsthereof.

In other aspects of the invention, compounds are screened for theability to specifically inhibit CB₁R/AT1R and/or CB₁R/A2aR heteromersover homodimers or monomers of the members comprising the heteromer towhich the composition binds (e.g., of A2aR, AT1R or CB₁R). Preferably,antagonist or inhibitory activity of a compound that specificallyinhibits a heteromer of the invention (e.g., the CB₁R/AT1R and/orCB₁R/A2aR heteromer) is about at least about 2-fold greater, morepreferably at least about 5-fold greater, and most preferably at leastabout 10-fold greater than the antagonist or inhibitory activity of thesame composition against homodimers or monomers of the memberscomprising the heteromer to which the composition binds (e.g., of A2aR,AT1R or CB₁R).

In certain embodiments of the invention, methods of screening for acompound useful for the treatment of liver disease are provided, whereinsuch methods include selecting compounds that inhibit endocannabinoidactivity in cells engineered to express CB₁R/AT1R heteromer, whereinsuch inhibition is greater than the inhibition of endocannabinoidactivity in cells engineered to express CB₁R alone or AT1R alone. Incertain aspects, this screening assay is carried out in HEK293 or CHOcells. In yet other aspects, the cells for use in the screening assaymay alternatively express the CB₁R/A2aR heteromer. Also Neuro2A cellsstably expressing AT1R or A2aR can be used in the screening assay sincethey express endogenous CB1R, and therefore will express CB1R/AT1R orCB1R/A2aR heteromers.

In one aspect of the invention, inhibition of endocannabinoid activityis determined by measuring ERK1/2 phosphorylation. Importantly, in theCB₁R/AT1R heteromeric complex, angiotensin II (Ang II), an agonist ofAT1R, can stimulate ERK1/2 phosphorylation when CB₁R is stimulated (withendocannabinoids/cannabinoid agonists, e.g., 2-AG). Thus, inhibition ofendocannabinoid activity is accompanied by a decrease in ERK1/2phosphorylation. ERK1/2 phosphorylation may be determined by Westernblot of whole cell lysates using a phospho-ERK (pERK) specific antibody.These methods are well known by those skilled in the art.

Preferably, the inhibition of endocannabinoid-mediated ERKphosphorylation in cells expressing the CB₁R/AT1R is about at leastabout 20 fold greater than the inhibition of endocannabinoid-mediatedERK phosphorylation in cells engineered to express CB₁R alone or AT1Ralone.

In certain embodiments of the present invention, methods of screeningfor a compound useful for the treatment of liver disease are provided.In some aspects, these methods include selecting compounds that decreaseintracellular cAMP levels in cells engineered to express a CB₁R/AT1Rheteromer. When AT1R heterodimerizes with CB₁R, it couples to inhibitoryG protein (Gi). Gi reduces the levels of intracellular cAMP. Thus,activation of AT1R within the CB₁R-AT1R complex leads to reduction inintracellular cAMP levels. Selective ligands for CB₁R-AT1R may beidentified by measuring the ability of the ligand or small molecule todecrease intracellular cAMP. In some embodiments of the invention, thedecrease in intracellular cAMP levels is greater than the decrease inintracellular cAMP levels in cells engineered to express CB₁R alone orAT1R alone. The levels of intracellular cAMP may be determined usingcommercially available detection kits, such as the one sold by Millipore(Billerica, Mass. 01821), Invitrogen (Carlsbad Calif. 92008), PerkinElmer (Waltham, Mass. 02451) etc.

A compound selected as a useful for treating liver disease in this assaywill preferably cause a decrease in intracellular cAMP levels in cellsexpressing the CB₁R/AT1R heteromer of at least about 5 fold greater thanthe decrease in intracellular cAMP levels in cells engineered to expressCB₁R alone or AT1R alone.

In certain aspects of the present invention, methods of screening for adiacylglycerol lipase (DAGL) inhibitor are provided. In certain aspects,these methods include selecting compounds that decrease ERK1/2phosphorylation in cells expressing CB₁R and another G-protein coupledreceptor (GPCR). In certain aspects, these receptors are only capable ofinducing ERK1/2 phosphorylation when the ligand for both the CB₁R andthe non-related GPCR are present together. Examples of the other GPCR ina complex with CB₁R are AT1R and A2aR. In certain embodiments, thereceptor ligands, e.g., 2-AG and angiotensin II (for the CB₁R/AT1Rheteromer) or 2-AG and adenosine (for the CB1R/A2aR heteromer) are mixedwith the screening composition and added to the test cells.

In some embodiments, the cells that may be used for this assay includebut are not limited to: Neuro2A, HEK293 cells, CHO cells and the like.

ERK1/2 phosphorylation may be determined by Western blot or any othermethod known in the art for determining the level of phosphorylatedprotein in a sample, such as a cell lysate. Such methods are describedin detail in Rozenfeld and Devi, 2007 (Receptor heteromerization leadsto a switch in signaling: beta-arrestin2-mediated ERK activation bymu-delta opioid receptor heteromers. Rozenfeld R, Devi L A. FASEB J.2007 August; 21(10):2455-65).

In other aspects, the DAGL screening assay may be conducted as ahigh-throughput screen of small molecules, such as drug-likepharmacophores.

In another preferred embodiment, methods (also referred to herein as“screening assays”) are provided for identifying candidate or testcompounds or agents which modulate the expression, function, activity ofthe heteromers, e.g. CB₁R-AT1R heteromers, CB₁R-A2aR heteromers.Compounds thus identified can be used to modulate the activity of targetgene products, e.g. CB₁R-AT1R heteromer, CB₁R-A2aR heteromer geneproducts, in a therapeutic protocol, to modulate the biological functionof the target gene product, or to identify compounds that disrupt normaltarget interactions.

After identifying a test compound or candidate agent as an agonistand/or an antagonist, the compound may then be used to treat subjectswith diseases and disorders associated with CB₁R-AT1R heteromers,CB₁R-A2aR heteromers.

Candidate agents also include numerous chemical classes, thoughtypically they are organic compounds including small organic compounds,nucleic acids including oligonucleotides, and peptides. Small organiccompounds suitably may have e.g. a molecular weight of more than about40 or 50 yet less than about 2,500. Candidate agents may comprisefunctional chemical groups that interact with proteins and/or DNA. Thesemolecules can be natural, e.g. from plants, fungus, bacteria etc., orcan be synthesized or synthetic.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckermann, R. N. et al.(1994)J. Med. Chem. 37:2678-85); spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the one-bead one-compound library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. AcadSci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad Sci. USA91:11422; Zuckermann et al. (1994). J. Med Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994)Angew. Chem. Int. Ed Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409),plasmids (Cull et al. (1992) Proc Nat'l Acad Sci USA 89:1865-1869) or onphage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382;Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

A prototype compound may be believed to have therapeutic activity on thebasis of any information available to the artisan. For example, aprototype compound may be believed to have therapeutic activity on thebasis of information contained in the Physician's Desk Reference. Inaddition, by way of non-limiting example, a compound may be believed tohave therapeutic activity on the basis of experience of a clinician,structure of the compound, structural activity relationship data, EC₅₀,assay data, IC₅₀ assay data, animal or clinical studies, or any otherbasis, or combination of such bases.

A therapeutically-active compound is a compound that has therapeuticactivity, including for example, the ability of a compound to induce aspecified response when administered to a subject or tested in vitro.Therapeutic activity includes treatment of a disease or condition,including both prophylactic and ameliorative treatment. Treatment of adisease or condition can include improvement of a disease or conditionby any amount, including prevention, amelioration, and elimination ofthe disease or condition. Therapeutic activity may be conducted againstany disease or condition, including in a preferred embodiment againsthepatitis virus, liver cancer, liver inflammation, cirrhosis or anycombination thereof. In order to determine therapeutic activity anymethod by which therapeutic activity of a compound may be evaluated canbe used. For example, both in vivo and in vitro methods can be used,including for example, clinical evaluation, EC₅₀, and IC₅₀ assays, anddose response curves.

Candidate compounds for use with an assay of the present invention oridentified by assays of the present invention as useful pharmacologicalagents can be pharmacological agents already known in the art orvariations thereof or can be compounds previously unknown to have anypharmacological activity. The candidate compounds can be naturallyoccurring or designed in the laboratory. Candidate compounds cancomprise a single diastereomer, more than one diastereomer, or a singleenantiomer, or more than one enantiomer.

Candidate compounds can be isolated, from microorganisms, animals orplants, for example, and can be produced recombinantly, or synthesizedby chemical methods known in the art. If desired, candidate compounds ofthe present invention can be obtained using any of the numerouscombinatorial library methods known in the art, including but notlimited to, biological libraries, spatially addressable parallel solidphase or solution phase libraries, synthetic library methods requiringdeconvolution, the “one-bead one-compound” library method, and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to polypeptide libraries. The other fourapproaches are applicable to polypeptide, non-peptide oligomer, or smallmolecule libraries of compounds and are preferred approaches in thepresent invention. See Lam, Anticancer Drug Des. 12: 145-167 (1997).

In an embodiment, the present invention provides a method of identifyinga candidate compound as a suitable prodrug. A suitable prodrug includesany prodrug that may be identified by the methods of the presentinvention. Any method apparent to the artisan may be used to identify acandidate compound as a suitable prodrug.

In another aspect, the present invention provides methods of screeningcandidate compounds for suitability as therapeutic agents. Screening forsuitability of therapeutic agents may include assessment of one, some ormany criteria relating to the compound that may affect the ability ofthe compound as a therapeutic agent. Factors such as, for example,efficacy, safety, efficiency, retention, localization, tissueselectivity, degradation, or intracellular persistence may beconsidered. In an embodiment, a method of screening candidate compoundsfor suitability as therapeutic agents is provided, where the methodcomprises providing a candidate compound identified as a suitableprodrug, determining the therapeutic activity of the candidate compound,and determining the intracellular persistence of the candidate compound.Intracellular persistence can be measured by any technique apparent tothe skilled artisan, such as for example by radioactive tracer, heavyisotope labeling, or LCMS.

In screening compounds for suitability as therapeutic agents,intracellular persistence of the candidate compound is evaluated. In apreferred embodiment, the agents are evaluated for their ability tomodulate the protein or peptide intracellular persistence may comprise,for example, evaluation of intracellular residence time or half-life inresponse to a candidate therapeutic agent. In a preferred embodiment,the half-life of a protein or peptide in the presence or absence of thecandidate therapeutic compound in human tissue is determined. Half-lifemay be determined in any tissue. Any technique known to the art workerfor determining intracellular persistence may be used in the presentinvention. By way of non-limiting example, persistence of a compound maybe measured by retention of a radiolabeled or dye labeled substance.

A further aspect of the present invention relates to methods ofinhibiting the activity of a condition or disease comprising the step oftreating a sample or subject believed to have a disease or conditionwith a prodrug identified by a compound of the invention. Compositionsof the invention act as identifiers for prodrugs that have therapeuticactivity against a disease or condition. In a preferred aspect,compositions of the invention act as identifiers for drugs that showtherapeutic activity against conditions including for example cancer.

In one embodiment, a screening assay is a cell-based assay in which acell expresses a CB₁R-AT1R heteromer or peptides thereof, or CB₁R-A2aRheteromers or peptides, CB₁R-AT1R-detectable marker construct or fusionprotein construct, for example, GST, luciferase fusion partners,isoforms or mutants thereof, which is contacted with a test compound,and the ability of the test compound to modulate the expression and/oractivity of the respective heteromers. Determining the ability of thetest compound to modulate can be accomplished by monitoring, forexample, immunoassays, blots, pull-down assays, etc, assays described indetail in the Examples section which follows. The cell, for example, canbe of mammalian origin, e.g., human.

In another preferred embodiment, the screening assay is ahigh-throughput screening assay.

In another preferred embodiment, soluble and/or membrane-bound forms ofisolated proteins, mutants or biologically active portions thereof, canbe used in the assays if desired. When membrane-bound forms of theprotein are used, it may be desirable to utilize a solubilizing agent.Examples of such solubilizing agents include non-ionic detergents suchas n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, TRITON™ X-100,TRITON™ X-114, THESIT™, Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

Cell-free assays can also be used and involve preparing a reactionmixture of the target gene or gene products thereof, and the testcompound under conditions and for a time sufficient to allow the twocomponents to interact and bind, thus forming a complex that can beremoved and/or detected.

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos, et al, U.S. Pat. No.4,868,103). A fluorophore label on the first, ‘donor’ molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the ‘donor’protein molecule may simply utilize the natural fluorescent energy oftryptophan residues. Labels are chosen that emit different wavelengthsof light, such that the ‘acceptor’ molecule label may be differentiatedfrom that of the ‘donor’. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. A FET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorimeter).

In another embodiment, determining the ability of a protein to bind or“dock” to a target molecule or docking site on a target molecule can beaccomplished using real-time Biomolecular Interaction Analysis (BIA)(see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem.63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol.5:699-705). “Surface plasmon resonance” or “BIA” detects biospecificinteractions in real time, without labeling any of the interactants(e.g., BLAcore). Changes in the mass at the binding surface (indicativeof a binding event) result in alterations of the refractive index oflight near the surface (the optical phenomenon of surface plasmonresonance (SPR)), resulting in a detectable signal which can be used asan indication of real-time reactions between biological molecules.

In one embodiment, the target product or the test substance is anchoredonto a solid phase. The target product/test compound complexes anchoredon the solid phase can be detected at the end of the reaction.Preferably, the target product can be anchored onto a solid surface, andthe test compound, (which is not anchored), can be labeled, eitherdirectly or indirectly, with detectable labels discussed herein.

Libraries:

Candidate agents may be also be obtained from a wide variety oflibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of e.g. bacterial, fungal and animal extracts are available orreadily produced.

Chemical Libraries:

Developments in combinatorial chemistry allow the rapid and economicalsynthesis of hundreds to thousands of discrete compounds. Thesecompounds are typically arrayed in moderate-sized libraries of smallmolecules designed for efficient screening. Combinatorial methods can beused to generate unbiased libraries suitable for the identification ofnovel compounds. In addition, smaller, less diverse libraries can begenerated that are descended from a single parent compound with apreviously determined biological activity. In either case, the lack ofefficient screening systems to specifically target therapeuticallyrelevant biological molecules produced by combinational chemistry suchas inhibitors of important enzymes hampers the optimal use of theseresources.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks,” such asreagents. For example, a linear combinatorial chemical library, such asa polypeptide library, is formed by combining a set of chemical buildingblocks (amino acids) in a large number of combinations, and potentiallyin every possible way, for a given compound length (i.e., the number ofamino acids in a polypeptide compound). Millions of chemical compoundscan be synthesized through such combinatorial mixing of chemicalbuilding blocks.

A “library” may comprise from 2 to 50,000,000 diverse member compounds.Preferably, a library comprises at least 48 diverse compounds,preferably 96 or more diverse compounds, more preferably 384 or morediverse compounds, more preferably, 10,000 or more diverse compounds,preferably more than 100,000 diverse members and most preferably morethan 1,000,000 diverse member compounds. By “diverse” it is meant thatgreater than 50% of the compounds in a library have chemical structuresthat are not identical to any other member of the library. Preferably,greater than 75% of the compounds in a library have chemical structuresthat are not identical to any other member of the collection, morepreferably greater than 90% and most preferably greater than about 99%.

The preparation of combinatorial chemical libraries is well known tothose of skill in the art. For reviews, see Thompson et al., Synthesisand application of small molecule libraries, Chem Rev 96:555-600, 1996;Kenan et al., Exploring molecular diversity with combinatorial shapelibraries, Trends Biochem Sci 19:57-64, 1994; Janda, Tagged versusuntagged libraries: methods for the generation and screening ofcombinatorial chemical libraries, Proc Natl Acad Sci USA. 91:10779-85,1994; Lebl et al., One-bead-one-structure combinatorial libraries,Biopolymers 37:177-98, 1995; Eichler et al., Peptide, peptidomimetic,and organic synthetic combinatorial libraries, Med Res Rev. 15:481-96,1995; Chabala, Solid-phase combinatorial chemistry and novel taggingmethods for identifying leads, Curr Opin Biotechnol. 6:632-9, 1995;Dolle, Discovery of enzyme inhibitors through combinatorial chemistry,Mol Divers. 2:223-36, 1997; Fauchere et al., Peptide and nonpeptide leaddiscovery using robotically synthesized soluble libraries, Can J.Physiol Pharmacol. 75:683-9, 1997; Eichler et al., Generation andutilization of synthetic combinatorial libraries, Mol Med Today 1:174-80, 1995; and Kay et al., Identification of enzyme inhibitors fromphage-displayed combinatorial peptide libraries, Comb Chem HighThroughput Screen 4:535-43, 2001.

Other chemistries for generating chemical diversity libraries can alsobe used. Such chemistries include, but are not limited to, peptoids (PCTPublication No. WO 91/19735); encoded peptides (PCT Publication WO93/20242); random bio-oligomers (PCT Publication No. WO 92/00091);benzodiazepines (U.S. Pat. No. 5,288,514); diversomers, such ashydantoins, benzodiazepines and dipeptides (Hobbs, et al., Proc. Nat.Acad. Sci. USA, 90:6909-6913 (1993)); vinylogous polypeptides (Hagihara,et al., J. Amer. Chem. Soc. 114:6568 (1992)); nonpeptidalpeptidomimetics with β-D-glucose scaffolding (Hirschmann, et al., J.Amer. Chem. Soc., 114:9217-9218 (1992)); analogous organic syntheses ofsmall compound libraries (Chen, et al., J. Amer. Chem. Soc., 116:2661(1994)); oligocarbamates (Cho, et al., Science, 261:1303 (1993)); and/orpeptidyl phosphonates (Campbell, et al., J. Org. Chem. 59:658 (1994));nucleic acid libraries (see, Ausubel, Berger and Sambrook, all supra);peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083);antibody libraries (see, e.g., Vaughn, et al., Nature Biotechnology,14(3):309-314 (1996) and PCT/US96/10287); carbohydrate libraries (see,e.g., Liang, et al., Science, 274:1520-1522 (1996) and U.S. Pat. No.5,593,853); small organic molecule libraries (see, e.g.,benzodiazepines, Baum C&E News, January 18, page 33 (1993); isoprenoids(U.S. Pat. No. 5,569,588); thiazolidinones and metathiazanones (U.S.Pat. No. 5,549,974); pyrrolidines (U.S. Pat. Nos. 5,525,735 and5,519,134); morpholino compounds (U.S. Pat. No. 5,506,337);benzodiazepines (U.S. Pat. No. 5,288,514); and the like.

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem. Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd., Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Bio sciences, Columbia, Md., etc.).

Small Molecules:

Small molecule test compounds can initially be members of an organic orinorganic chemical library. The agonists and antagonists of the presentinvention can be small molecules. The term “small molecule” as usedherein refers to compounds, chemicals, small molecules, small moleculeinhibitors, or other factors that are useful for antagonizing oragonizing the target signaling complexes set forth in the invention(e.g. CB₁R/AT1R heteromers, CB₁R/A2aR heteromers, CB₁R only whendimerized with AT1R or A2aR, AT1R only when dimerized with CB₁R, or A2aRonly when dimerized with CB₁R).

These “small molecules” can be small organic or inorganic molecules ofmolecular weight below about 3,000 Daltons. The small molecules can benatural products or members of a combinatorial chemistry library. A setof diverse molecules should be used to cover a variety of functions suchas charge, aromaticity, hydrogen bonding, flexibility, size, length ofside chain, hydrophobicity, and rigidity. Combinatorial techniquessuitable for synthesizing small molecules are known in the art, e.g., asexemplified by Obrecht and Villalgordo, Solid-Supported Combinatorialand Parallel Synthesis of Small-Molecular-Weight Compound Libraries,Pergamon-Elsevier Science Limited (1998), and include those such as the“split and pool” or “parallel” synthesis techniques, solid-phase andsolution-phase techniques, and encoding techniques (see, for example,Czarnik, Curr. Opin. Chem. Bio., 1:60 (1997). In addition, a number ofsmall molecule libraries are commercially available.

Diverse libraries of small molecule inhibitors can be generated. Thecompounds of the present invention, particularly libraries of variantshaving various representative classes of substituents, are amenable tocombinatorial chemistry and other parallel synthesis schemes (see, forexample, PCT WO 94/08051). The result is that large libraries of relatedcompounds, e.g., a variegated library of potential receptor-heteromerantagonists, can be screened rapidly in high-throughput assays toidentify potential lead compounds, as well as to refine the specificity,toxicity, and/or cytotoxic-kinetic profile of a lead compound.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules such as the subjectantagonists. See, for example, Blondelle et al. (1995) Trends Anal.Chem. 14: 83; the Affymax U.S. Pat. Nos. 5,359,115 and 5,362,899: theEllman U.S. Pat. No. 5,288,514: the Still et al. PCT publication WO94/08051; Chen et al. (1994) JACS 116: 2661: Kerr et al. (1993) JACS115: 252; PCT publications WO92/10092, WO93/09668 and WO91/07087; andthe Lerner et al. PCT publication WO93/20242). Accordingly, a variety oflibraries on the order of about 100 to 1,000,000 or more diversomers ofthe subject antagonists can be synthesized and screened for a particularactivity or property. These methods are described in detail in U.S. Pat.No. 6,916,821.

Data and Analysis:

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, forexample Setubal and Meidanis et al., Introduction to ComputationalBiology Methods (PWS Publishing Company, Boston, 1997); Salzberg,Searles, Kasif, (Ed.), Computational Methods in Molecular Biology,(Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:Application in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2nd ed., 2001). See U.S. Pat.No. 6,420,108.

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention relates to embodiments that includemethods for providing information over networks such as the Internet.

Compositions

In certain aspects, the present invention provides compositionscomprising monoclonal antibodies or small compounds. For example,compositions comprising a CB₁R/AT₁R specific monoclonal antibody areprovided. Administration may be achieved by any suitable method.

The compounds of the invention can be formulated for administration inany convenient way for use in human or veterinary medicine. Themonoclonal antibodies of the invention may be incorporated intoliposomes, microemulsions, micelles, unilamellar or multilamellarvesicles, erythrocyte ghosts or spheroblasts. In one embodiment, themonoclonal antibody and/or small compounds of the invention can bedelivered in a vesicle, including as a liposome (see Langer, Science,1990; 249:1527-1533; Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss:New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.).

In yet another embodiment, monoclonal antibodies or small compounds ofthe invention can be delivered in a controlled release form. Forexample, one or more small compounds may be administered in a polymermatrix such as poly (lactide-co-glycolide) (PLGA), in a microsphere orliposome implanted subcutaneously, or by another mode of delivery (see,Cao et al., 1999, Biomaterials, February; 20(4):329-39). Another aspectof delivery includes the suspension in an alginate hydrogel.

The present invention also provides pharmaceutical formulations ordosage forms for administration to mammals in need thereof.

When formulated in a pharmaceutical composition, the compounds of thepresent invention can be admixed with a pharmaceutically acceptablecarrier or excipient. The phrase “pharmaceutically acceptable” refers tomolecular entities and compositions that are “generally regarded assafe”, e.g., that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehicleswith which the compound is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water or aqueoussaline solutions and aqueous dextrose and glycerol solutions arepreferably employed as carriers, particularly for injectable solutions.Alternatively, the carrier can be a solid dosage from carrier, includingbut not limited to one or more of a binder (for compressed pills), anencapsulating agent, a flavorant, and a colorant. Suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E.W. Martin.

Administration

The compounds and formulations of the present invention can beadministered topically, parenterally, orally, by inhalation, as asuppository, or by other methods known in the art. The term “parenteral”includes injection (for example, intravenous, epidural, intrathecal,intramuscular, intraluminal, intratracheal or subcutaneous). Thecompounds of the present invention may also be administered using atransdermal patch. In a specific embodiment, the compounds of thepresent invention are administered via an oral dosage form.

By “transdermal patch” is meant a system capable of delivery of a drugto a patient via the skin, or any suitable external surface, includingmucosal membranes, such as those found inside the mouth. Such deliverysystems generally comprise a flexible backing, an adhesive and a drugretaining matrix, the backing protecting the adhesive and matrix and theadhesive holding the whole on the skin of the patient. On contact withthe skin, the drug-retaining matrix delivers drug to the skin,permitting the drug to pass through the skin into the patient's system.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Administration of the compounds of the invention may be once a day,twice a day, or more often, but frequency may be decreased during amaintenance phase of the disease or disorder, e.g., once every second orthird day instead of every day or twice a day. The dose and theadministration frequency will depend on the clinical signs, whichconfirm maintenance of the remission phase, with the reduction orabsence of at least one or more preferably more than one clinical signsof the acute phase known to the person skilled in the art. Moregenerally, dose and frequency will depend in part on recession ofpathological signs and clinical and subclinical symptoms of a diseasecondition or disorder contemplated for treatment with the presentcompounds.

The antagonists described herein can be used to treat or prevent liverdiseases, obesity and other metabolic disorders.

The benefit to an individual to be treated is either statisticallysignificant or at least perceptible to the patient or to the physician.

It will be appreciated that the amount of the antagonists of theinvention required for use in treatment will vary with the route ofadministration, the nature of the condition for which treatment isrequired, and the age, body weight and condition of the patient, andwill be ultimately at the discretion of the attendant physician orveterinarian. These compositions will typically contain an effectiveamount of the compounds of the invention, alone or in combination withan effective amount of any other active material, e.g., those describedabove. Preliminary doses can be determined according to animal tests,and the scaling of dosages for human administration can be performedaccording to art-accepted practices.

Keeping the above description in mind, typical dosages of monoclonalantibody may range from about 5 to about 400 mg/h. In certainembodiments, a patient may receive, for example, one or more doses.

An “agonist” is a molecule which activates a certain type of receptor.For example, endocannabinoids act as agonists when they excitecannabinoid receptors. An example of an agonist of the present inventionis 2 arachidonoylglycerol (2-AG), which is an agonist of CB₁R. AT1R:AngII. CB1R: ACEA: Potent, highly selective CB1 agonist; ACPA: Potent,selective CB1 agonist; Arvani: Potent CB1 and TRPV1 agonist. Alsoanandamide transport inhibitor; (±)-CP 47497: Potent CB1 agonist; DEA:Endogenous CB1 agonist; Leelamine hydrochloride: CB1 agonist;(R)-(+)-Methanandamid: Potent and selective CB1 agonist; Hu210, 2-AG:non-selective CB1 agonists. A2aR: CGS 21680 hydrochloride: A2A agonist;CV 180: Non-selective adenosine A2 receptor agonist; adenosine:Non-selective adenosine A2 receptor agonist.

By contrast, an “antagonist” is a molecule which prevents or reduces theeffects exerted by an agonist on a receptor. An example of an antagonistof the present invention is a monoclonal antibody (mAb) which isgenerated against the CB₁R/AT1R heteromer complex using a subtractiveimmunization strategy. The mAb preferentially recognizes the CB₁R/AT1Rheteromer over CB₁R alone or AT1R alone and blocks or decreases theactivity of the CB₁R/AT1R heteromer. The term “therapeutic index” refersto the therapeutic index (TI) of a drug, defined as LD₅₀/ED₅₀. Otherexamples of antagonists of CB1R, AT1R and A2AR include, for example,Rimonabant (SR141716), CP-945,598, Losartan (Merck, Whitehouse Station,N.J.) etc. A2aR antagonists: SCH 442416: Very selective, high affinityA2A antagonist; SCH 58261: Potent, highly selective A2A antagonist; ZM241385: Potent, highly selective A2A antagonist (TOCRIS, Ellisville, Mo.63021). CB1R antagonists: AM 251: Potent CB1 antagonist. Also GPR55agonist; LY 320135: Selective CB1 receptor antagonist/inverse agonist;0-2050 CB1 silent antagonist. AT1R antagonists: EMD 66684: Potent,selective non-peptide AT1 antagonist; ZD 7155 hydrochloride: Selectivenon-peptide AT1 antagonist.

Preferred antagonists are those that provide a reduction of activationby the ligand of at least 10%, and more preferably at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or even at least 99% at aconcentration of the antagonist, for example, of 1 μg/ml, 10 μg/ml, 100μg/ml, 500 μg/ml, 1 mg/ml, 10 mg/ml, or 100 mg/ml. The percentageantagonism represents the percentage decrease in activity ofendocannabinoids, e.g., 2-AR, in a comparison of assays in the presenceand absence of the antagonist. Any combination of the above mentioneddegrees of percentage antagonism and concentration of antagonist may beused to define an antagonist of the invention, with greater antagonismat lower concentrations being preferred.

Antibodies

In a preferred embodiment, a compound comprises an antibody or fragmentsthereof. Preferably, the antibody is a monoclonal antibody and isspecific for CB₁R-AT1R heteromers or CB₁R-A2aR heteromers as compared tocontrol receptors, for example, CB₁R monomers, CB₁R homodimers, AT1Rmonomers, AT1R homodimers, A2aR monomers, or A2aR homodimers. Suitableantibodies or antibody fragments may be also be polyclonal, orrecombinant. Examples of useful fragments include separate heavy chains,light chains, scFv, Fab, F(ab′)₂, Fabc, and Fv fragments. Typically, theantibodies, fragments, or similar binding agents bind a specific antigenwith an affinity of at least 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹.

In certain embodiments, a monoclonal antibody that is specific for aCB1-AT1 heteromer is the antibody designated mAb A108. Monoclonalantibody mAb A108 may be used in any of the compositions and methodsdescribed herein and is specifically contemplated for use in suchcompositions and methods.

Various host animals selected, e.g., from pigs, cows, horses, rabbits,goats, sheep, rats, or mice, can be immunized with a partially orsubstantially purified epitope peptide identified above, or with apeptide homolog, fusion protein, peptide fragment, analog or derivativethereof. For example, the peptides for immunization can be conjugated toan immune potentiator (such as keyhole limpet hemocyanin (KLH), serumalbumin, bovine thyroglobulin, or soybean trypsin inhibitor using abifunctional or derivatizing agent) to enhance antigen-specificresponses. An adjuvant can be used to enhance antibody production.Examples of useful adjuvants include without limitation oil emulsions(e.g., Freund's adjuvant), saponins (e.g., saponin derivative QS-21),aluminium or calcium salts (e.g., alum), non-ionic block polymersurfactants, lipopolysaccharides (e.g., lipopolysaccharide-derived MPL),mycobacteria, tetanus toxoid, MF59 microemulsion, lipid-particleimmuno-stimulating complexes (ISCOMs), CpG oligonucleotides and manyothers (see, e.g., Gupta et al., 1993, Vaccine, 11:293-306). Polyclonalantibodies can be obtained and isolated from the serum of an immunizedanimal.

In a preferred embodiment, an antibody specifically binds to CB₁R-AT1Rheteromers, proteins, peptides, fragments, variants, orthologs, alleles,isoforms, splice variants, derivatives or mutants thereof as compared toa control receptor, for example, CB₁R monomers, CB₁R homodimers, AT1Rmonomers, AT1R homodimers.

In a preferred embodiment, an antibody specifically binds to CB₁R-A2aRheteromers, proteins, peptides, variants, orthologs, alleles, isoforms,splice variants, fragments, derivatives or mutants thereof, as comparedto a control receptor. For example, CB₁R monomers, CB₁R homodimers, A2aRmonomers, A2aR homodimers.

Anyone of the antibodies will have applications for research use (ex:Western blots, immunohistochemistry, flow cytometry, imaging, ELISA) aswell as clinical applications for prognostics and potential monitoringof therapeutic regimens using automated antibody-based platforms andpathological analysis of patient samples.

Anti-CB₁R/GPCR heteromer antibodies or antigen-binding portions of thepresent invention can have agonist-like and/or antagonist-likeproperties on CB₁R/GPCR heteromers of the invention, such as CB₁R/AT1Rand CB₁R/A2aR heteromers. These antibodies are therefore considered tobe agonists or antagonists of their target. Preferably, anti-CB₁R/GPCRheteromer antibodies of the invention have an antagonist-like effect(i.e., are antagonists of their target heteromer). Such an antibody orantigen-binding portion would, upon binding to its antigen, exhibit atleast one antagonist-like effect. In one embodiment, the antibody orantigen-binding portion of the present invention would exhibittherapeutic effectiveness like an antagonist of CB₁R (e.g., Rimonabant(SR141716)) and also have the ability to reduce undesirable effects ofagonists of CB₁R/GPCR heteromer (e.g., 2-AG for CB₁R/AT1R heteromer)resulting from setting off cascades of second messenger responses (i.e.,cAMP increase.). Preferably, the antibody or antigen-binding portion ofthe present invention exhibits greater therapeutic effectiveness than anantagonist of CB₁R (e.g., Rimonabant (SR141716)), which has undesirableside effects. Such greater therapeutic effectiveness, e.g., for thetreatment of liver disease, is achieved by virtue of the specificity ofthe antibodies of the invention for the CB₁R/AT1R or CB₁R/A2aR heteromercomplex rather than either monomer of the complex alone.

Radiolabeling:

In another preferred embodiment, the antibody of the invention can beradiolabeled. Uses include therapeutic and imaging for diagnosticpurposes. The label may be a radioactive atom, an enzyme, or achromophore moiety. Methods for labeling antibodies have been described,for example, by Hunter and Greenwood, Nature, 144:945 (1962) and byDavid et al. Biochemistry 13:1014-1021 (1974). Additional methods forlabeling antibodies have been described in U.S. Pat. Nos. 3,940,475 and3,645,090. Methods for labeling oligonucleotide probes have beendescribed, for example, by Leary et al. Proc. Natl. Acad. Sci. USA(1983) 80:4045; Renz and Kurz, Nucl. Acids Res. (1984) 12:3435;Richardson and Gumport, Nucl. Acids Res. (1983) 11:6167; Smith et al.Nucl. Acids Res. (1985) 13:2399; and Meinkoth and Wahl, Anal. Biochem.(1984) 138:267.

The label may be radioactive. Some examples of useful radioactive labelsinclude ³²P, ¹²⁵I, ¹³¹I, and ³H. Use of radioactive labels have beendescribed in U.K. 2,034,323, U.S. Pat. Nos. 4,358,535, and 4,302,204.

Some examples of non-radioactive labels include enzymes, chromophores,atoms and molecules detectable by electron microscopy, and metal ionsdetectable by their magnetic properties.

Some useful enzymatic labels include enzymes that cause a detectablechange in a substrate. Some useful enzymes and their substrates include,for example, horseradish peroxidase (pyrogallol and o-phenylenediamine),β-galactosidase (fluorescein β-D-galactopyranoside), and alkalinephosphatase (5-bromo-4-chloro-3-indolyl phosphate/nitro bluetetrazolium). The use of enzymatic labels has been described in U.K.2,019,404, EP 63,879, and by Rotman, Proc. Natl. Acad Sci. USA, 47,1981-1991 (1961).

Useful chromophores include, for example, fluorescent, chemiluminescent,and bioluminescent molecules, as well as dyes. Some specificchromophores useful in the present invention include, for example,fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone,luminol.

The labels may be conjugated to the antibody or nucleotide probe bymethods that are well known in the art. The labels may be directlyattached through a functional group on the probe. The probe eithercontains or can be caused to contain such a functional group. Someexamples of suitable functional groups include, for example, amino,carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate.Alternatively, labels such as enzymes and chromophores may be conjugatedto the antibodies or nucleotides by means of coupling agents, such asdialdehydes, carbodiimides, dimaleimides, and the like.

The label may also be conjugated to the probe by means of a ligandattached to the probe by a method described above and a receptor forthat ligand attached to the label. Any of the known ligand-receptorcombinations is suitable. Some suitable ligand-receptor pairs include,for example, biotin-avidin or biotin-streptavidin, and antibody-antigen.

In another preferred embodiment, the antibody molecules of the inventioncan be used for imaging. In imaging uses, the complexes are labeled sothat they can be detected outside the body. Typical labels areradioisotopes, usually ones with short half-lives. The usual imagingradioisotopes, such as ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ^(99m)TC, ¹⁸⁶Re, ¹⁸⁸Re,⁶⁴Cu, ⁶⁷Cu, ²¹²Bi, ²¹³Bi, ⁶⁷Ga, ⁹⁰Y, ¹¹¹In, ¹⁸F, ³H, ¹⁴C, ³⁵S or ³²P canbe used. Nuclear magnetic resonance (NMR) imaging enhancers, such asgadolinium-153, can also be used to label the complex for detection byNMR. Methods and reagents for performing the labeling, either in thepolynucleotide or in the protein moiety, are considered known in theart.

Aptamers:

CB1R-AT1R or CB1R-A2aR specific molecules can be in the form ofaptamers. “Aptamers” are DNA or RNA molecules that have been selectedfrom random pools based on their ability to bind other molecules. Theaptamer binds specifically to a target molecule wherein the nucleic acidmolecule has sequence that comprises a sequence recognized by the targetmolecule in its natural setting. Alternately, an aptamer can be anucleic acid molecule that binds to a target molecule wherein the targetmolecule does not naturally bind to a nucleic acid. The target moleculecan be any molecule of interest. For example, the aptamer can be used tobind to a ligand-binding domain of a protein, thereby preventinginteraction of the naturally occurring ligand with the protein. This isa non-limiting example and those in the art will recognize that otherembodiments can be readily generated using techniques generally known inthe art (see, e.g., Gold et al., Annu. Rev. Biochem. 64:763, 1995; Brodyand Gold, J. Biotechnol. 74:5, 2000; Sun, Curr. Opin. Mol. Ther. 2:100,2000; Kusser, J. Biotechnol. 74:27, 2000; Hermann and Patel, Science287:820, 2000; and Jayasena, Clinical Chem. 45:1628, 1999).

As used herein, the term “aptamer” or “selected nucleic acid bindingspecies” shall include non-modified or chemically modified RNA or DNA.The method of selection may be by, but is not limited to, affinitychromatography and the method of amplification by reverse transcription(RT) or polymerase chain reaction (PCR).

The affinity of aptamers for their target proteins is typically in thenanomolar range, but can be as low as the picomolar range. That is K_(D)is typically 1 μM to 500 nM, more typically from 1 μM to 100 nM.Apatmers having an affinity of K_(D) in the range of 1 μM to 10 nM arealso useful.

Anti-CB₁R/GPCR Heteromer Antibodies

Subtractive Immunization:

For induction of tolerance to immunogenic epitopes in HEK-293 membranes,female balb/c mice (6-8 weeks old, 25-35 g body weight) were injectedintraperitoneally (i.p.) with 5 mg Neuro-2A membranes and 15 min laterwith cyclophosphamide (100 mg/kg body weight, i.p.). Thecyclophosphamide injection was repeated after 24 and 48 h respectively.Mice were bled every 15 days and antibody titers checked by ELISAagainst Neuro-2A membranes. This protocol was repeated at 2 weekintervals until stable background titers were obtained with Neuro-2Amembranes. Mice were then given an i.p. injection of membranes fromNeuro-2A cells expressing AT1 angiotensin receptors (5 mg) in completeFreund's adjuvant. Booster i.p. injections of Neuro-2A cells expressingAT1 angiotensin receptors were administered every 15 days. Antibodytiters were checked by ELISA against Neuro2A membranes fromuntransfected cells and from cells expressing AT1 receptors. Spleensfrom animals giving a high titer with Neuro-2A cells expressing AT1angiotensin receptors were fused with SP-20 myeloma cells to generatemonoclonal antibodies as described (Gupta et al., JBC 282:5116, 2007).Clones secreting monoclonal antibodies were screened by ELISA againstuntransfected Neur-2A membranes, and Neuro-2A cells expressing AT1angiotensin receptors using 1:10 hybridoma supernatant and 1:500 horseradish peroxidase labeled anti-mouse IgG. Hybridoma supernatant frompositive clones was concentrated using Centricon 10 and stored at aconcentration of 10 μg protein/μl.

The present invention thus provides isolated monoclonal antibodies andantigen-binding portions thereof that specifically bind to CB1R/AT1R orCB1R/A2aR heteromers. In certain embodiments of the invention, theantibody compositions of the invention exhibit antagonist-like activityon their heteromer targets (CB1R/AT1R or CB1R/A2aR heteromer).

In other aspects of the invention, gene expression is determined usingpolymerase chain reaction (PCR). PCR is a well-known method that allowsexponential amplification of short DNA sequences (usually 100 to 600bases) within a longer double stranded DNA molecule.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, recombinant DNA,immunology, cell biology and other related techniques within the skillof the art. See, e.g., Sambrook et al., (2001) Molecular Cloning: ALaboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: ColdSpring Harbor, N.Y.; Sambrook et al., (1989) Molecular Cloning: ALaboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press: ColdSpring Harbor, N.Y.; Ausubel et al., eds. (2005) Current Protocols inMolecular Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Bonifacinoet al., eds. (2005) Current Protocols in Cell Biology. John Wiley andSons, Inc.: Hoboken, N.J.; Coligan et al., eds. (2005) Current Protocolsin Immunology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coico et al.,eds. (2005) Current Protocols in Microbiology, John Wiley and Sons,Inc.: Hoboken, N.J.; Coligan et al., eds. (2005) Current Protocols inProtein Science, John Wiley and Sons, Inc.: Hoboken, N.J.; Enna et al.,eds. (2005) Current Protocols in Pharmacology John Wiley and Sons, Inc.:Hoboken, N.J.; Hames et al., eds. (1999) Protein Expression: A PracticalApproach. Oxford University Press: Oxford; Freshney (2000) Culture ofAnimal Cells: A Manual of Basic Technique. 4th ed. Wiley-Liss; amongothers. The Current Protocols listed above are updated several timesevery year.

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. Numerous changes to the disclosedembodiments can be made in accordance with the disclosure herein withoutdeparting from the spirit or scope of the invention. Thus, the breadthand scope of the present invention should not be limited by any of theabove described embodiments.

All documents mentioned herein are incorporated herein by reference. Allpublications and patent documents cited in this application areincorporated by reference for all purposes to the same extent as if eachindividual publication or patent document were so individually denoted.By their citation of various references in this document, Applicants donot admit any particular reference is “prior art” to their invention.Embodiments of inventive compositions and methods are illustrated in thefollowing examples.

EXAMPLES

The present invention is described further below in working exampleswhich are intended to further describe the invention without limitingthe scope therein.

Materials and Methods

Ligand Binding Assay: Stably transfected cells were seeded in 12-wellplates for 24 h. Cells were washed briefly in cold wash buffer (25 mMTris-HCl, pH 7.4) containing 140 mM NaCl, 5 mM MgCl₂, and 0.1% bovineserum albumin (BSA). Binding experiments were performed at 48 C andinitiated by the addition of 50 000 cpm of w3Hx-Ang II (69 Ci/mmol)(Amersham Pharmacia Biotech) in the presence of varying amounts ofunlabeled Ang II (Sigma, St. Louis, Mo., USA) or compound of interest asa competitor in a 0.5-ml volume of binding buffer w25 mM Tris-HClbuffer, pH 7.4, including 5 mM MgCl₂, 0.1% BSA and 100 mg/ml bacitracin(Sigma). Then, 24 h after cold incubation, cells were washed twice in 1ml of wash buffer and lysed using 0.5 ml of lysis buffer (0.1 M NaOHcontaining 0.5% of SDS). Receptor-bound radioactivity was determined ona β-counter (Packard Instruments, Downer Grove, Ill., USA) and thebinding data (IC₅₀ values) were analyzed using GraphPad software(GraphPad Software, San Diego, Calif., USA). To obtain Kd, the Cheng andPrusoff (1973) (Biochem Pharmacol. 1973 Dec. 1; 22(23):3099-108)equation was used and Bmax was calculated according to DeBlasi et al.(1989) Trends Pharmacol Sci. 10(6):227-9. All measurements are carriedout in triplicate and results are expressed as mean±SEM. Statisticalsignificance is evaluated using Student's t-test.

Example 1: CB₁R and AT1R Form a Complex in Activated HSCs and CB₁RActivity Controls Ang II-Mediated pERK Materials and Methods

HSCs from ethanol treated rats were generated in the laboratory of Dr.Natalia Nieto: Rats (300 g female Sprague-Dawley, N=10/group) were fedthe control or ethanol Lieber-DeCarli diets for 8 months (Lieber andDeCarli, 1989). Animals received humane care according to the criteriaoutlined in the Guide for Care and Use of Laboratory Animals. Detailsregarding pathology of the liver of the control and alcohol fed rats aredescribed in (Cubero and Nieto, 2008; Urtasun et al., 2009). Briefly,Hematoxylin and eosin staining showed microvesicular and macrovesicularsteatosis in livers from ethanol-fed rats; and transaminases andnonesterified fatty acids were elevated twofold and sixfoldrespectively, in the ethanol-fed rats.

Generation of Ethanol-Treated Rats: Chronic Alcohol Feeding Model. Rats(300 g female Sprague-Dawley, N=10/group) were fed the control orethanol Lieber-DeCarli diets for 8 months (Lieber C S, DeCarli L M.Liquid diet technique of ethanol administration: 1989 update. AlcoholAlcohol 1989; 24: 197-211). Animals received human care according to thecriteria outlined in the Guide for Care and Use of Laboratory Animals.ALT, AST, ethanol, and non-esterified fatty acids were assayed usingkits from Thermo Electron Corporation (Waltham, Mass.), Sigma (St.Louis, Mo.), and Wako Chemicals (Richmond, Va.), respectively. H&Estaining and TEM sections were prepared according to standardmethodology and evaluated by a liver pathologist.

Isolation of Hepatic Stellate Cells: These were isolated as describedbefore. (Friedman, S. L. Hepatic stellate cells: protean,multifunctional, and enigmatic cells of the liver. Physiol Rev 88,125-72 (2008); Teixeira-Clerc, F. et al. CB1 cannabinoid receptorantagonism: a new strategy for the treatment of liver fibrosis. Nat Med12, 671-6 (2006)).

Western Blots and Immunoprecipitation: Western blot and phospho-ERKAssays were carried out as described in the case of experiments withNeuro2A cells (Rozenfeld and Devi, 2008). For experiments with HSCs,freshly plated cells were stimulated for 10 minutes with AngII in thepresence or absence of SR141716 and THL (as indicated). Phospho ERK andERK were detected with rabbit monoclonal anti-phospho-p44/42 MAPK(anti-pERK, 1:1000) and mouse monoclonal anti-p44/42 MAPK (anti-ERK,1:1,000) antibodies. Both blotting and imaging with the Odyssey imagingsystem (LI-COR, Lincoln, Nebr.) were performed following themanufacturer's protocols. The secondary antibodies that were usedincluded IRDye 680-labeled anti-rabbit antibody, IRDye 800-labeledanti-mouse and anti goat antibodies (1:10,000).

Results

CB₁R protein levels exhibited marked upregulation (˜7-fold) in HSCs fromethanol treated rats (eHSCs) compared to HSCs from control rats (cHSCs)(FIG. 1A), indicating that, consistent with other experimental models ofliver injury, chronic ethanol treatment leads to CB₁R upregulation inHSCs. Activation of the eHSCs was also supported by a substantialincrease in the levels of α-smooth muscle actin (α-SMA), a marker of HSCactivation (FIG. 1A). However, the expression of a profibrogenicreceptor, AT1R, which was also expressed in cHSCs, displayed only aslight increase (<2-fold) in activated HSCs (FIG. 1A).

To investigate the influence of upregulated CB₁R on resident AT1R, thedirect interaction between the two receptors was examined. It was foundthat CB₁R can be detected in AT1R-immunoprecipitate in activated but notin control HSCs (FIG. 1B), indicating that the two receptors werepresent in an interacting complex in activated HSCs.

The interaction between CB1R and A2aR was also examined, sincestimulation of this receptor activates mitogenic pathways leading to theexpression of fibrosis markers (Chan E S, et al., (2006) Br J Pharmacol148:1144-1155; Che J, Chan E S, Cronstein B N (2007). Mol Pharmacol72:1626-1636.) A2aR was constitutively expressed in HSCs, displayingonly a slight increase in its levels (˜15%) in activated HSCs (FIG. 1C).In these cells, A2aR associated with CB1R since co-immunoprecipitationlead to the isolation of the two receptors in an interacting complex(FIG. 1D).

It was examined if CB₁R interacts with AT1R and A2aR in vivo. Chronictreatment with CCl₄ lead to upregulation of CB₁R, A2aR, and to a lesserextent, of AT1R expression in mice liver (FIG. 1E).Co-immunoprecipitation experiments using livers from vehicle and CCl₄treated mice showed that CB1R interacted with AT1R and with A2aR infibrotic but not in control livers (FIG. 1F). Similarly CB1R was inducedin human cirrhotic livers (FIG. 1G) and co-immunoprecipitationexperiments showed that CB₁R interacted with A2aR in human livers.Further, there was enhanced association of CB₁R with AT1R and with A2aRin livers from cirrhotic patients as compared to control patients (FIG.1H).

It was examined if, in the CB₁R-AT1R heteromer, AT1R signaling wasaffected by CB₁R. Angiotensin II (Ang II)-mediated mitogenic signalingwas focused on because of the implication of this pathway infibrosis^(4,5). Ang II treatment led to a marked enhancement of pERKlevels in eHSCs compared to cHSCs. This increase in Ang II-mediatedsignaling was due to interactions with CB₁R since treatment with a CB₁Rspecific antagonist prevented Ang II-mediated ERK phosphorylation ineHSCs but not cHSCs (FIG. 2A). The role of endocannabinoids was furtherexamined in AT1R signaling by blocking 2-arachidonoyl glycerol (2-AG)formation using the diacylglycerol lipase (DAGL) inhibitortetrahydrolipstatin (THL). THL treatment decreased Ang II-mediated ERKphosphorylation in eHSCs, but not cHSCs (FIG. 2B). This, taken with thefinding that CB₁R antagonist blocked Ang II-mediated pERK indicated thatin activated HSCs, Ang II-mediated signaling was controlled byendocannabinoid-dependent CB₁R activity.

Similarly, it was found that the A2aR agonist-mediated enhancement ofpERK levels was blocked by the CB₁R antagonist (FIG. 2C). These resultsindicated that CB₁R activity controlled the signaling and profibrogenicpotential of at least two GPCRs involved in HSC activation and liverfibrosis.

Example 2: Characterization of the CB₁R-AT1R Complex and CB₁R RegulatesAng-II Mediated Increase in pERK Materials and Methods

Immunofluorescence and Confocal Microscopy Immunofluorescence andConfocal Microscopy was carried out as described (Rozenfeld and Devi,2008). Slides were visualized with a Leica TCS SP5 confocal microscope.Images were acquired with an x63/1.32 PL APO objective lens, andanalyzed in sequential scanning mode.

Generation of HEK293-Transfected Cells and Neuro2A Cell Line StablyExpressing AT1R. These cell lines were generated using standardprocedure of transfection and antibiotic selection of expressing cells.Screening of positive cells was carried out by immunostaining of thereceptors to evaluate the homogeneity of expression in the clones.

Western Blots and Immunoprecipitation Coimmunoprecipitation and Westernblotting—Cells were lysed for 1 h in lysis buffer (1% Triton, 150 mMNaCl, 1 mM EDTA, 1 mM EGTA, and 50 mM Tris-Cl, pH 7.4) containingprotease inhibitor cocktail (Sigma). For immunoprecipitation, celllysates containing 400-600 gig of protein was incubated with theanti-AT1R or anti-A2aR antibody/protein A/C agarose complex overnight at4° C. The beads were washed three times with lysis buffer and once withthe same buffer without detergent. Proteins were eluted in 60 μL of 2×Laemmli buffer containing 1% 2-mercaptoethanol. Proteins were resolvedby 10% SDS-PAGE, and subjected to Western blotting as described(Rozenfeld and Devi, 2008). Western blot and phospho-ERK Assays werecarried out as described in the case of experiments with Neuro2A cells(Rozenfeld and Devi, 2008). For experiments with HSCs, freshly platedcells were stimulated for 10 minutes with AngII in the presence orabsence of SR141716 and THL (as indicated). Phospho ERK and ERK weredetected with rabbit monoclonal anti-phospho-p44/42 MAPK (anti-pERK,1:1000) and mouse monoclonal anti-p44/42 MAPK (anti-ERK, 1:1,000)antibodies. Both blotting and imaging with the Odyssey imaging system(LI-COR, Lincoln, Nebr.) were performed following the manufacturer'sprotocols. The secondary antibodies that were used included IRDye680-labeled anti-rabbit antibody, IRDye 800-labeled anti-mouse and antigoat antibodies (1:10,000).

Results

In order to clearly define the molecular mechanism underlying thefunctional interaction between CB₁R and AT1R, recombinant systems wereused. In transfected HEK293 cells, AT1R expressed alone was located atthe plasma membrane, whereas CB₁R was located in intracellular vesicles,as previously reported⁶ (immunofluorescence data not shown). Whenco-expressed with AT1R, CB₁R localized primarily to the plasma membranealong with AT1R (immunofluorescence data not shown). In these cells,stimulation with Ang II lead to a greater increase in pERK (as comparedto cells expressing AT1R alone). Furthermore, in these cells, modulationof CB₁R activity by the antagonist SR141716 (SR), or by inhibiting theendocannabinoid 2-AG production by the administration of THL, lead to amarked decrease in Ang II-mediated signaling (FIG. 3A). These resultsare in agreement with the results obtained in the eHSCs and show thatirrespective of the cell system, CB₁R affects AT1R properties in asimilar fashion. Thus, the recombinant system is a useful model toexplore the mechanisms of CB R-AT1R interactions.

A neuroblastoma cell line (Neuro2A) that contained endogenous CB₁Rreceptors was used, and a Neuro2A cell line was generated stablyexpressing AT1R. In these cells, CB₁R and AT1R were colocalized and AT1Rcould be detected in CB₁R-immunoprecipitate, indicating the associationof CB₁R-AT1R within a complex (immunofluorescence data not shown. Ang IIstimulation lead to a dose-dependent rapid and transient enhancement ofpERK levels (FIGS. 9A and 9B) and RNAi-mediated CB₁R down-regulationlead to a dramatic decrease in this effect (FIG. 3B; FIGS. 9D & 10A),reminiscent of what was observed with cHSCs. Blocking CB₁R using thespecific antagonist SR141716 also blocked >80% of Ang II-mediated pERK(FIG. 2D; FIG. 10B). Blocking the production of the endocannabinoid 2-AGwith THL lead to a loss of Ang II-mediated signaling (FIG. 2E); this wasreversed by addition of Hu210 (a CB1R agonist, commercially availablefrom Tocris Bioscience, Ellisville, Mo. 63021) or of exogenous 2-AG,(FIGS. 2E and H). These results, taken with the fact that stimulatingCB₁R with a non-signaling concentration of CB₁R agonist potentiates AT1Rsignaling (FIG. 2F; FIG. 10C) confirm that AT1R signaling is controlledby CB₁R activity in the context of the CB R-AT1R heteromer.

Example 3: CB₁R Switches AT1R Coupling to Gαi Materials and Methods

Dominant Negative Gαq Construct

The construct is available at cDNA.org. Details are described below: TheQ209L and D277N mutations were introduced into the human G protein alphaq subunit (GNAOQ00000) via the Quickchange mutagenesis kit (Stratagene).The mutations confer a preference for xanthine nucleotide binding.Insert size=1085 bp.

RNAi-mediated CB₁R and βarrestin-2 downregulation. Plasmid and siRNAtransfections were carried out as described (Rozenfeld and Devi, 2007FASEB J. 2007 August; 21(10):2455-65).

Western Blots and Immunoprecipitation. Coimmunoprecipitation and Westernblotting—Cells were lysed for 1 h in lysis buffer (1% Triton, 150 mMNaCl, 1 mM EDTA, 1 mM EGTA, and 50 mM Tris-Cl, pH 7.4) containingprotease inhibitor cocktail (Sigma). For immunoprecipitation, celllysates containing 400-600 gig of protein was incubated with theanti-AT1R or anti-A2aR antibody/protein A/C agarose complex overnight at4° C. The beads were washed three times with lysis buffer and once withthe same buffer without detergent. Proteins were eluted in 60 μL of 2×Laemmli buffer containing 1% 2-mercaptoethanol. Proteins were resolvedby 10% SDS-PAGE, and subjected to Western blotting as described(Rozenfeld and Devi, 2008). Western blot and phospho-ERK Assays werecarried out as described in the case of experiments with Neuro2A cells(Rozenfeld and Devi, 2008). For experiments with HSCs, freshly platedcells were stimulated for 10 minutes with AngII in the presence orabsence of SR141716 and THL (as indicated). Phospho ERK and ERK weredetected with rabbit monoclonal anti-phospho-p44/42 MAPK (anti-pERK,1:1000) and mouse monoclonal anti-p44/42 MAPK (anti-ERK, 1:1,000)antibodies. Both blotting and imaging with the Odyssey imaging system(LI-COR, Lincoln, Nebr.) were performed following the manufacturer'sprotocols. The secondary antibodies that were used included IRDye680-labeled anti-rabbit antibody, IRDye 800-labeled anti-mouse and antigoat antibodies (1:10,000).

Results

To delineate the novel pathway of the heteromer-mediated signaling, thenature of the G protein coupling to AT1R was investigated. Expression ofa dominant negative Gαq construct affected AT1R signaling only underconditions of CB₁R down-regulation (FIG. 2F; FIG. 12A) indicating a lossof AT1R coupling to Gαq. Next, it was examined if there was a shift incoupling to Gαi. Treatment with pertussis toxin (PTX) (which blocks Gαi)markedly inhibited AT1R signaling in cells co-expressing CB₁R and AT1Rand this was reduced upon down-regulation of CB₁R expression (achievedby RNAi) (FIG. 21; FIG. 12B). It was also found that forskolin(Sigma-Aldrich, St. Louis, Mo. 63103) treatment (which interferes withGαi-mediated signaling by preventing efficient inhibition of adenylylcyclase) decreased AT1R-mediated signaling in the CB₁R-AT1R expressingcells (FIG. 12C), supporting a switch in G protein coupling facilitatedby heteromerization. This switch in coupling of AT1R indicated apossible mechanism of G protein sequestration by the Gαi coupledreceptor CB₁R^(7,8).

Finally, it was also found that heteromerization lead to changes in thenature of the interactions between AT1R and βarrestin-2. In contrast tothe established contribution of pβarrestin-2 in AT1R mitogenicsignaling⁹, it was found that down-regulation of pβarrestin-2 (by RNAi)did not inhibit AT1R-mediated pERK in the CB₁R-AT1R expressing cells(FIG. 12D) confirming the change in the nature of coupling of AT1R byheteromerization with CB₁R.

Example 4: CB₁R and A2aR Form a Complex in Activated HSCs, and CB₁RActivity Controls A2aR-Mediated pERK Materials and Methods

Western Blots and Immunoprecipitation. Coimmunoprecipitation and Westernblotting—Cells were lysed for 1 h in lysis buffer (1% Triton, 150 mMNaCl, 1 mM EDTA, 1 mM EGTA, and 50 mM Tris-Cl, pH 7.4) containingprotease inhibitor cocktail (Sigma). For immunoprecipitation, celllysates containing 400-600 μg of protein was incubated with theanti-AT1R or anti-A2aR antibody/protein A/C agarose complex overnight at4° C. The beads were washed three times with lysis buffer and once withthe same buffer without detergent. Proteins were eluted in 60 μL of 2×Laemmli buffer containing 1% 2-mercaptoethanol. Proteins were resolvedby 10% SDS-PAGE, and subjected to Western blotting as described(Rozenfeld and Devi, 2008). Western blot and phospho-ERK Assays werecarried out as described in the case of experiments with Neuro2A cells(Rozenfeld and Devi, 2008). For experiments with HSCs, freshly platedcells were stimulated for 10 minutes with AngII (Tocris Bioscience,Ellisville, Mo. 63021) in the presence or absence of SR141716 (TocrisBioscience, Ellisville, Mo. 63021) and THL (F. Hoffmann-La Roche Ltd,Basel, Switzerland) as indicated. Phospho ERK and ERK were detected withrabbit monoclonal anti-phospho-p44/42 MAPK (anti-pERK, 1:1000) and mousemonoclonal anti-p44/42 MAPK (anti-ERK, 1:1,000) antibodies. Bothblotting and imaging with the Odyssey imaging system (LI-COR, Lincoln,Nebr.) were performed following the manufacturer's protocols. Thesecondary antibodies that were used included IRDye 680-labeledanti-rabbit antibody, IRDye 800-labeled anti-mouse and anti goatantibodies (1:10,000).

Results

In addition to CB₁R and AT1R, several GPCRs have been shown to beinvolved in the development of fibrosis indicating that modulation ofsignaling by heteromerization with CB₁R can be extended to otherreceptors. A2aR was focused on, since stimulation of this receptoractivates mitogenic pathways leading to the expression of fibrosismarkers^(3, 10). A2aR is constitutively expressed in HSCs, displayingonly a slight increase in its levels (˜15%) in activated HSCs (FIG. 1C).In these cells, A2aR associates with CB₁R since co-immunoprecipitationleads to the isolation of the two receptors in an interacting complex(FIG. 1D). More importantly, the A2aR agonist-mediated enhancement ofpERK levels (via the A2aR agonist CGS21680 (Tocris Bioscience,Ellisville, Mo. 63021) is blocked by the CB₁R antagonist SR141716 (SR)(Tocris Bioscience, Ellisville, Mo. 63021) (FIG. 3A).

These results indicated that CB₁R activity controls the signaling andprofibrogenic potential of at least two GPCRs involved in HSC activationand liver fibrosis.

Example 5: CB₁R Antagonist Prevents Ang II-Mediated Fibrogenic Responsein eHSCs Materials and Methods

Reverse Transcription and Real-Time PCR. Total RNA was isolated from3×10⁸ HSCs using the TRIzol method (Invitrogen, Carlsbad, Calif., USA).RNA (1.0 μg) was reverse-transcribed in 20 μl of buffer containing 50 μMoligo(dT)20, 25 mM MgCl₂, 0.1 M dithiothreitol, 40 U/μl RNaseOUT, and200 U/μl SuperScript III RT for 50 min at 50° C. The reaction wasstopped by incubating the samples at 85° C. for 5 min, and 40 μl ofnuclease-free water was added. Real-time PCR was performed by using theBrilliant SYBR Green QPCR Master Mix. The PCR template source was either30 ng of first-strand cDNA or purified DNA standard. Primer sequencesused to amplify the desired cDNA are detailed in Table 2. Amplificationwas performed with a spectrofluorometric thermal cycler (Stratagene).After an initial denaturation step at 95° C. for 10 min, amplificationwas performed using 40 cycles of denaturation (95° C. for 30 s),annealing (56° C. for 1 min), and extension (72° C. for 1 min). Tostandardize mRNA levels, we amplified GAPDH, a housekeeping gene, as aninternal control. Gene expression was normalized by calculating theratio between the number of cDNA copies of collagen type I, type III,TGF-β, α-SMA and that of GAPDH. For analysis of gene expression, afterreverse transcription, the number of copies of mRNA for the indicatedtranscripts were determined by real-time PCR. Data were normalized toGAPDH mRNA and are expressed as the mean±SD (n=3 in quadruplicate).

CB₁R antagonists. SR141716: eHSCs were stimulated with Ang II in theabsence or presence of SR141716 (SR; Tocris Bioscience, Ellisville, Mo.63021) for 4 hours before the RNA was harvested.

CGS21680 (CGS)- eHSCs were stimulated with CGS (Tocris Bioscience,Ellisville, Mo. 63021) in the absence or presence of SR for 6 hoursbefore the RNA was harvested. In order to determine the CGS-mediatedfibrogenic response in mouse fibrotic liver, CCl₄- or vehicle-treatedmice were injected intraperitoneally (i.p.) with vehicle, CGS, or CGS+AM251 (AM, CB₁R antagonist). After 6 hours, the mice were sacrificed,their liver quickly removed and the RNA harvested.

Results

The role of CB₁R complexes were explored in the regulation of theexpression of profibrogenic markers in liver fibrosis. It was found thatstimulation of AT1R or A2aR leads to an increase in expression of α-SMA,TGFβ, pro-collagen α III and pro-collagen α I by as much as 120-160%over baseline in activated HSCs; this increase can be completely blockedby concomitant treatment with the CB₁R antagonist (FIGS. 4A and 4B)indicating that the profibrogenic potential of these receptors is underthe control of CB₁R activity.

Example 6: Anti-CB₁R/AT1R Heteromer Monoclonal Antibody Materials andMethods

A subtractive immunization strategy was used to generate antibodies thatselectively recognize the endogenous CB₁R/AT1R heteromer but do notrecognize either the CB₁R or AT1R receptors alone. Mice were first madetolerant to unwanted epitopes on membrane proteins by the simultaneousadministration of Neuro2A cell membranes (which endogenously expressCB₁R and cyclophosphamide).

Subtractive Immunization:

For induction of tolerance to immunogenic epitopes in HEK-293 membranes,female balb/c mice (6-8 weeks old, 25-35 g body weight) were injectedintraperitoneally (i.p.) with 5 mg Neuro-2A membranes and 15 min laterwith cyclophosphamide (100 mg/kg body weight, i.p.). Thecyclophosphamide injection was repeated after 24 and 48 h respectively.Mice were bled every 15 days and antibody titers checked by ELISAagainst Neuro-2A membranes. This protocol was repeated at 2 weekintervals until stable background titers were obtained with Neuro-2Amembranes. Mice were then given an i.p. injection of membranes fromNeuro-2A cells expressing AT1 angiotensin receptors (5 mg) in completeFreund's adjuvant. Booster i.p. injections of Neuro-2A cells expressingAT1 angiotensin receptors were administered every 15 days. Antibodytiters were checked by ELISA against Neuro2A membranes fromuntransfected cells and from cells expressing AT1 receptors. Spleensfrom animals giving a high titer with Neuro-2A cells expressing AT1angiotensin receptors were fused with SP-20 myeloma cells to generatemonoclonal antibodies as described (Gupta et al., JBC 282:5116, 2007).Clones secreting monoclonal antibodies were screened by ELISA againstuntransfected Neuro-2A membranes, and Neuro-2A cells expressing AT1angiotensin receptors using 1:10 hybridoma supernatant and 1:500 horseradish peroxidase labeled anti-mouse IgG. Hybridoma supernatant frompositive clones was concentrated using Centricon 10 and stored at aconcentration of 10 μg protein/μl.

This results in the destruction of antibody-generating activated Bcells. Once a low titer to Neuro-2A membrane proteins was achieved[Gupta et al., J Biol Chem. 2007 Dec. 21; 282(51):36797-807], mice wereimmunized with Neuro-2A membranes expressing AT1R receptors [Gomes etal., Methods Mol Med. 84:157, 2003].

The antibody titer was monitored after booster injections and once ahigh titer was obtained, the spleens of these mice were used to generatemonoclonal antibodies (mAbs).

Results

TABLE 1 Number of clones expressing CB1-AT1 heteromer specificantibodies. Positive Titer CB1-AT1 AT1 CB1 Between 2-3 1 zero zeroBetween 1-2 18 zero 1 Between 0.5-1 31 5 1

The supernatants from the hybridoma clones were screened with Neuro-2Amembranes alone, membranes from cells expressing only AT1 receptors, andmembranes from cells co-expressing both CB₁R and AT1R receptors. Thisled to the identification of a number of antibody-secreting clones thatgave a high titer with membranes co-expressing CB₁R-AT1R receptors[Gupta et al., JBC 282:5116, 2007].

The specificity of the AT1R-CB1R antibody for cells expressing AT1R andCB1R, but not for cells expressing either of these receptors alone isdemonstrated by results shown in FIGS. 8A-E. AT1R-CB1R antibody wastested for binding of AT1R-CB1R in Neuro2A, Neuro2A-AT1R, andNeuro2A-AT1R in which CB1R expression was downregulated by RNAi using anenzyme-linked immunoabsorbent assay (ELISA). ELISA assay indicated thepresence of a specific AT1R-CB1R epitope detected only in Neuro2A-AT1R(FIG. 8A). AT1R-CB1R antibody was screened against membranes from HEK293cells coexpressing AT1R and CB1R, or membranes from cells expressingonly AT1R or CB1R. ELISA assay indicated the presence of a specificAT1R-CB1R epitope detected only in AT1R-CB1R expressing cells (FIG. 8B).There was no signal with membranes from cells coexpressing CB1R withCB2R, DOR, MOR or KOR, or cells expressing DOR with KOR or MOR (FIG.8C).

The specificity of the antibody was further characterized using cellsthat expressed different ratios of CB1R and AT1R. Maximal recognition bythe heteromer antibody was observed when CB1R and AT1R were expressed atsimilar levels, but not when expressed at 1:5 or 5:1 ratios) (FIG. 8D).AT1R-CB1R was also used to measure the AT1R-CB1R immunoreactivity fromactivated (eHSCs) and control (cHSCs) hepatic stellate cells. ELISAassay indicated the presence of a specific AT1R-CB1R epitope detectedonly in activated but not in quiescent HSCs (FIG. 8E).

The ability of the heteromer-specific monoclonal antibody to selectivelyinhibit signaling by CB₁R-AT1R heteromers was examined. CB1R-AT1Rmediated activity was selectively blocked by heteromer specificantibody. Heteromer-selective antibody significantly blocked AT1Ractivity mediated increases in ERK phosphorylation (FIGS. 8F and 8G).These results demonstrate that the monoclonal antibody against theAT1R-CB1R heteromer blocks receptor signaling only in the context of theheteromer but not when the receptors are expressed alone.

Example 7: CB₁R Activity Controls Other GPCR-Mediated Pro-FibrogenicGene Expression and Properties in Animal Models of Fibrosis Materialsand Methods

Murine model of liver fibrosis and human tissues. Wild-type C54b16 micewere treated with i.p. injections of CCl₄ (2 mL/kg CCl4 mixed withmineral oil; 10% v/v) or mineral oil, 3 times a week for 8 weeks (n=5-6per group). There were no deaths in any of the groups. 48 hours afterthe last CCl4 injection, mice were injected i.p. with vehicle (DMSO 4%in saline), CGS21680 (2 mg/kg) or AM251 (Tocris Bioscience, Ellisville,Mo. 63021) (2 mg/kg)+CGS21680. Six hours after the injections, mice weresacrificed by CO2 narcosis. Aliquots of liver tissue (50-100 mg) wereflash frozen and kept at −80° C. until RNA isolation. The tissue wasminced and homogenized in TRIzol reagent, and RNA was extractedaccording to the manufacturer's instructions and then dissolved insterile DEPC water and stored at −80° C. RT-PCR was performed asdescribed:

Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction.Total RNA was isolated from 3×10⁸ HSCs using the TRIzol method(Invitrogen, Carlsbad, Calif., USA). RNA (1.0 μg) wasreverse-transcribed in 20 μl of buffer containing 50 μM oligo(dT)20, 25mM MgCl2, 0.1 M dithiothreitol, 40 U/μl RNaseOUT, and 200 U/μlSuperScript III RT for 50 min at 50° C. The reaction was stopped byincubating the samples at 85° C. for 5 min, and 40 μl of nuclease-freewater was added. Real-time PCR was performed by using the Brilliant SYBRGreen QPCR Master Mix. The PCR template source was either 30 ng offirst-strand cDNA or purified DNA standard. Primer sequences used toamplify the desired cDNA are detailed in Table 2. Amplification wasperformed with a spectrofluorometric thermal cycler (Stratagene). Afteran initial denaturation step at 95° C. for 10 min, amplification wasperformed using 40 cycles of denaturation (95° C. for 30 s), annealing(56° C. for 1 min), and extension (72° C. for 1 min). To standardizemRNA levels, GAPDH, a housekeeping gene, was amplified as an internalcontrol. Gene expression was normalized by calculating the ratio betweenthe number of cDNA copies of TGF-β and α-SMA and that of GAPDH.

TABLE 2 Collagen (α1) Sense 5′ CGA CTA AGT TGG AGG GAA CGG TC 319 (I) 3′(SEQ ID NO: 1) Antisense 5′ TGG CAT GTT GCT AGG CAC GAC 3′(SEQ ID NO: 2) Collagen (α1) Sense 5′ CGA GGT GAC AGA GGT GAA AGA 3′ 336(III) (SEQ ID NO: 3) Antisense 5′ AAC CCA GTA TTC TCC GCT CTT 3′(SEQ ID NO: 4) TGF-β1 Sense 5′ TAT AGC AAC AAT TCC TGG CG 3′ 162(SEQ ID NO: 5) Antisense 5′ TGC TGT CAC AAG AGC AGT G 3′ (SEQ ID NO: 6)GAPDH Sense 5′ TGT GTC TGT CGT GGA TCT GA 3′  76 (SEQ ID NO: 7)Antisense 5′ CCT GCT TCA CCA CCT TCT TGA 3′ (SEQ ID NO: 8) α-SMASense: TCC TCC CTG GAG AAG AGC TAC (SEQ ID NO: 9)Antisense: TAT AGG TGG TTT CGT GGA TGC (SEQ ID NO: 10)

Results

In livers from CCl₄ treated mice, upregulated expression ofprofibrogenic genes was further increased by acute injection with theA2aR agonist CGS (FIG. 4C). Co-administration of the CB1R antagonistAM251 with CGS prevented CGS-induced profibrogenic gene expression. CGStreatment did not elevate the expression of profibrogenic genes innon-fibrotic mouse livers (from vehicle-treated mice), supporting thenotion that A2aR exhibits profibrogenic properties only in fibroticlivers. The study herein study shows that specific changes occurring inactivated HSCs, such as CB₁R upregulation can cause A2aR to beprofibrogenic. Altogether, these results indicate that in activatedHSCs, CB₁R activity controls other GPCR-mediated profibrogenic geneexpression and properties, and this can be observed in vivo, in animalmodels of liver fibrosis.

Example 8: Chronic Alcohol Feeding Model Materials and Methods

Rats (300 g female Sprague-Dawley, N=10/group) were fed the control orethanol Lieber-DeCarli diets for 8 months (See Lieber C S, DeCarli L M.Liquid diet technique of ethanol administration: 1989 update. AlcoholAlcohol 1989; 24: 197-211). Animals received human care according to thecriteria outlined in the Guide for Care and Use of Laboratory Animals.ALT, AST, ethanol, and non-esterified fatty acids were assayed usingkits from Thermo Electron Corporation (Waltham, Mass.), Sigma (St.Louis, Mo.), and Wako Chemicals (Richmond, Va.), respectively. H&Estaining and TEM sections were prepared according to standardmethodology and evaluated by a liver pathologist.

Details regarding pathology of the liver of the control and alcohol fedrats are available. See, Cubero F J, Nieto N. Ethanol and arachidonicacid synergize to activate Kupffer cells and modulate the fibrogenicresponse via tumor necrosis factor alpha, reduced glutathione, andtransforming growth factor beta-dependent mechanisms; Hepatology. 2008December; 48(6):2027-39; Urtasun R, Cubero F J, Vera M, Nieto N.Reactive nitrogen species switch on early extracellular matrixremodeling via induction of MMP1 and TNFalpha. Gastroenterology. 2009;136: 1410-22.

Results

Ultrastructural analysis depicting micro- and macrovesicular steatosis,vacuolization, and electron dense mitochondria in livers from ethanolfed rats (FIG. 12A, right) compared to livers from control rats (FIG.12A, left) (magnification=2500×). FIG. 12B shows light micrographs fromHSC isolated from control and ethanol-fed rats. Details regardingpathology of the liver of the control and alcohol fed rats are describedin (Cubero and Nieto, 2008; Urtasun et al., 2009). Briefly, Hematoxylinand eosin staining showed microvesicular and macrovesicular steatosis inlivers from ethanol-fed rats; and transaminases and nonesterified fattyacids were elevated twofold and sixfold respectively, in the ethanol-fedrats.

Example 9: Methods of Screening for DAG Lipase Inhibitors Materials andMethods

Neuro2A cells expressing endogenous CB₁R and recombinant AT1R werepretreated with vehicle or THL (2 μM) for 3 hours in serum free mediaand stimulated with vehicle (CTL=control) or with the receptor ligand(angiotensin II) for 3 minutes. Cells were lysed and the cell lysateswere probed for the levels of pERK1/2 and ERK1/2 by Western blot, usinganti ERK and pERK from Cell Signaling Technology (Danvers, Mass. 01923).To assess the effect of THL on Ang II-mediated ERK phosphorylation inNeuro2A-AT1 cells, cells were pretreated with THL (1 μM) for two hours,then they were stimulated with 10 nM Ang II in the presence or absenceof 2-AG (100 nM), for 3 minutes. ERK phosphorylation was assessed byWestern blot using antibodies to pERK and ERK (n=3-5). To set up a highthroughput screening assay, 24 h after plating, the cells were starvedfor 2 hours, then incubated with vehicle (DMSO) or with THL (1 μM) for 2hours, then stimulated with Ang II (10 nM) for 3 minutes. The cells wereprocessed for infrared detection of pERK.

Results

A receptor complex system was set up involving dimerization of CB₁R withAT1R. This system exhibits the unique property that neither CB₁R nor theassociated receptor (AT1R) is able to signal through the ERK1/2 pathwayupon stimulation with its cognate agonist. However, co-stimulation witha combination of the agonists for both receptors leads to ERK1/2phosphorylation. Inhibition of the production of 2-AG using the lipaseinhibitor THL prevents 2-AG-mediated basal stimulation of CB₁R. In theseconditions, there is a marked decrease in ERK1/2 phosphorylation uponagonist stimulation of the associated receptor (FIG. 5).

Using the same conditions of inhibition of 2-AG production with THL, theassociated receptor was stimulated with a constant concentration ofagonist, and stimulated CB₁R by concomitant treatment with increasingdoses of 2-AG. A concentration-dependent 2-AG-mediated ERK1/2phosphorylation was obtained. See FIGS. 6 and 7.

These data indicate that the receptor complex system can be used toscreen for inhibitor of DAG lipase involved in the production of 2-AG:In basal conditions (in the absence of inhibition), the concentration of2-AG is sufficient to allow ERK1/2 phosphorylation after stimulation ofthe associated receptor. In the case of inhibition of the DAG lipase,the absence of 2-AG prevents ERK1/2 phosphorylation induced by treatmentwith the ligand of the associated receptor. The same experiments wereconducted as described above in 96-well plates. Similar results wereobtained, indicating that this method can be used for screening ofinhibitors of DAG lipase in a high-throughput scale.

For high-throughput screening, test concentrations of compounds may bebetween 10 and 100 mM. A cut-off of 100 mM may be used in selectingcompounds active in the primary screen and counter-screening assays.First, the compounds are tested to determine if they can inhibit theactivity of DAGL in the cell based assay described above using acompound concentration of 10 mM. A screening compound is selected forfurther testing as a DAGL inhibitor if it mediates at least a 70% ormore decrease in Ang II-mediated ERK phosphorylation compared to thepositive control. In all assays, THL is used as a positive control.Libraries of small molecules and drug-like pharmacophores may be usedERK phosphorylation is determined by Western blot using an antibodyspecific for phosphorylated ERK.

This DAGL-inhibitor screen is specific for CB₁R activation, it issensitive and is based on a simple assay (ERK1/2 phosphorylation) thatcan be easily adapted to high throughput. In addition, it does notinvolve measurement of lipid levels.

DISCUSSION

Preventing the expression or activation of CB₁Rs attenuates thedevelopment of fibrosis². Here a novel mechanism is described for thepivotal role of CB₁R in liver disease, involving the formation offibrosis-specific receptor heteromers consisting of upregulated CB₁Rwith resident fibrogenic G protein coupled receptors (GPCRs) inactivated HSCs. Specifically, there is enhanced association of CB₁R withangiotensin II receptor (AT1R) and adenosine 2a receptor (A2aR) inactivated HSCs and in liver from CCl₄ treated mice and cirrhotic humanpatient; within these complexes, AT1R and A2aR activity-mediatedmitogenic signaling events that lead to profibrogenic effects arecompletely under the control of CB₁R activity. Disrupting CB₁Rexpression or activity leads to a dramatic decrease in angiotensinII-mediated fibrogenic signaling, demonstrating that CB₁R controls AT1Rsignaling in activated HSCs. Similarly, association of CB₁R with theadenosine 2a receptor (A2aR), another receptor involved in fibrosis 3also leads to the control of adenosine signaling by CB₁R andprofibrogenic gene expression by CB₁R in activated HSCs and in anexperimental model of liver fibrosis. Taken together, these resultsdemonstrate a general mechanism of signal hijacking by CB₁R in activatedHSCs and demonstrate the generation of context-dependent GPCR heteromersthat occur only in a pathological state, which in turn leads tomodification of signaling that contribute to the disease. Furthermore,the data herein define these receptor heteromers (CB₁R-AT1R andCB₁R-A2aR) as disease-specific drug targets for the treatment of liverfibrosis.

In this study, a mechanism was identified for the contribution of CB₁Ractivity in liver fibrosis, through heteromerization with other GPCRs.Upregulated CB₁R in activated HSCs forms complexes with AT1R and A2aRand this leads to a shift in G protein coupling and increased signaling,which ultimately results in enhancing the profibrogenic activity ofthese associating receptors.

The role of CB₁R in liver disease is poorly understood. The findingherein, that CB₁R uses other receptors to amplify their profibrogenicsignaling sheds light on one mechanism by which CB₁R regulates liverfibrosis. This, together with the possibility of auto and paracrinetransactivation of CB₁R by AT1R^(11, 12) demonstrates a loop ofamplification of cannabinoid/angiotensin and cannabinoid/adenosinesignaling by several mechanisms converging towards upregulation ofprofibrogenic stimulation through the endocannabinoid system.

Another important finding in this study is that the basal CB₁R activityis sufficient to enhance AT1R and A2aR responses. This demonstrates thatlow or no basal activity of CB₁R can decrease the fibrogenic potentialof Ang II and adenosine, underscoring a particularly important role forthe endocannabinoid tone in the maintenance of fibrosis. Hence,inhibiting the enzymes involved in endocannabinoid production representsnovel therapy for the treatment of liver fibrosis.

Abnormal production of 2-AG is a hallmark of a number of diseases,including liver fibrosis. Blocking the activation of the cannabinoidsystem by using cannabinoid receptor antagonists validates thecannabinoid system as an outstanding drug target. Because blocking ofCB₁R alone leads to adverse CNS effects, an alternative strategy toblock the activation of the cannabinoid system was identified herein;namely, a screening method to identify inhibitors of the production ofthe abnormally high levels of endocannabinoid ligands. The fewinhibitors of this pathway that have been identified are not orallyactive, limiting their use as pharmaceutically-acceptable drugs. Priorto this invention, there was no method suitable for the high throughputscreening (HTS) of DAGL inhibitors. This is due to the lipidic nature ofthe substrates/products of DAGL that requires labor-intensive methods ofanalysis. The two main assays to measure DAGL activity currently are (i)measuring the conversion of sn-1-[3H]oleoyl-2-arachidonoylglycerol to[3H]oleic acid and 2-AG using thin layer chromatography; and (ii)measuring endocannabinoid levels by liquid chromatography coupled tomass spectrometry.

In summary, a novel, fast cell-based assay that measuresendocannabinoid-mediated cell signaling is described. This assay takesadvantage of the finding that CB1R heteromerization mediates changes insignaling. This assay is useful to screen for and identify DAGLinhibitors, which are valuable therapeutic tools for the treatment ofliver diseases, such as cirrhosis and alcoholic and non-alcoholicfibrosis, obesity, metabolic disease.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all values are approximate, and areprovided for description.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

LITERATURE CITED

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1.-17. (canceled)
 18. A method of screening for a candidate compounduseful for the treatment of a liver disease, the method comprising: a)contacting the compound with a CB1R/A2aR heteromer, and with at leastone control receptor selected from the group consisting of a CB1Rmonomer, a CB1R homodimer, an A2aR monomer, and an A2aR homodimer, b)measuring inhibition of an activity of the CB1R/A2aR heteromer and ofthe control receptor in the presence of the compound, and c) selectingthe compound which preferentially inhibits the activity of the CB1R/A2aRheteromer as compared to the control receptor, as a candidate compounduseful for the treatment of the liver disease.
 19. The method of claim18, wherein the candidate compound inhibits the activity of theCB1R/A2aR heteromer at least about 5-fold greater than the activity ofthe control receptor.
 20. The method of claim 18, wherein the activityis determined by measuring phosphorylation of extracellularsignal-regulated kinase-1 (ERK1) or extracellular signal-regulatedkinase-2 (ERK2).
 21. The method of claim 18, wherein the compound is amonoclonal antibody.
 22. The method of claim 18, wherein the compound isa small molecule. 23-31. (canceled)
 32. A method of screening for acompound useful for the treatment of a liver disease, the methodcomprising: a) contacting the compound with a cell expressing a type Icannabinoid receptor (CB1R)/adenosine 2a receptor (A2aR) heteromer, andwith a control cell expressing CB1R alone or A2aR alone; b) measuringinhibition of endocannabinoid activity in the cell expressing CB1R/A2aRheteromer and in the control cell in the presence of the compound, andc) selecting the compound which preferentially inhibits endocannabinoidactivity in the cell expressing the CB1R/A2aR heteromer as compared tothe control cell, as the candidate compound useful for the treatment ofthe liver disease.
 33. The method of claim 32, wherein the inhibition ofendocannabinoid activity is determined by measuring adenosine-mediatedERK1/2 phosphorylation.
 34. The method of claim 32, wherein theinhibition of endocannabinoid activity in the cell expressing theCB1R/A2aR heteromer is at least about 5-fold greater than the inhibitionof endocannabinoid activity in the control cell.
 35. The method of claim32, wherein the cell expressing the CB1R/A2aR heteromer, CB1R alone, orA2aR alone is HEK293 or CHO cell. 36.-46. (canceled)